Integrated monolithic microfabricated dispensing nozzle and liquid chromatography-electrospray system and method

ABSTRACT

A droplet/electrospray device and a liquid chromatography-electrospray system are disclosed. The droplet/electrospray device comprises a substrate defining a channel between an entrance orifice on an injection surface and an exit orifice on an ejection surface, a nozzle defined by a portion recessed from the ejection surface surrounding the exit orifice, and an electrode for application of an electric potential to the substrate to optimize and generate droplets or an electrospray. A plurality of these electrospray devices can be used in the form of an array of miniaturized nozzles. The liquid chromatography-electrospray device comprises a separation substrate defining an introduction channel between an entrance orifice and a reservoir and a separation channel between the reservoir and an exit orifice, the separation channel being populated with separation posts perpendicular to the fluid flow. A cover substrate is bonded to the separation substrate to enclose the reservoir and the separation channel adjacent the cover substrate. The exit orifice of the liquid chromatography device is homogeneously interfaced with the entrance orifice of the electrospray device to form an integrated single system. Procedures for fabrication of the electrospray devices of the present invention are also disclosed.

This application is a division of U.S. patent application Ser. No.09/468,535, Dec. 20, 1999, now U.S. Pat. No. 6,633,031 which claims thebenefit of U.S. Provisional Patent Application Ser. No. 60/122,972,filed Mar. 2, 1999.

FIELD OF THE INVENTION

The present invention relates generally to an integrated miniaturizedfluidic system fabricated using microelectromechanical systems (MEMS)technology, particularly to an integrated monolithic microfabricateddispensing nozzle capable of dispensing fluids in the form of dropletsor as an electrospray of the fluid.

BACKGROUND OF THE INVENTION

New trends in drug discovery and development are creating new demands onanalytical techniques. For example, combinatorial chemistry is oftenemployed to discover new lead compounds, or to create variations of alead compound. Combinatorial chemistry techniques can generate thousandsof compounds (combinatorial libraries) in a relatively short time (onthe order of days to weeks). Testing such a large number of compoundsfor biological activity in a timely and efficient manner requireshigh-throughput screening methods which allow rapid evaluation of thecharacteristics of each candidate compound.

The compounds in combinatorial libraries are often tested simultaneouslyagainst a molecular target. For example, an enzyme assay employing acolorimetric measurement may be run in a 96-well plate. An aliquot ofenzyme in each well is combined with tens of compounds. An effectiveenzyme inhibitor will prevent development of color due to the normalenzyme reaction, allowing for rapid spectroscopic (or visual) evaluationof assay results. If ten compounds are present in each well, 960compounds can be screened in the entire plate, and one hundred thousandcompounds can be screened in 105 plates, allowing for rapid andautomated biological screening of the compounds.

The quality of the combinatorial library and the compounds containedtherein is used to assess the validity of the biological screening data.Confirmation that the correct molecular weight is identified for eachcompound or a statistically relevant number of compounds along with ameasure of compound purity are two important measures of the quality ofa combinatorial library. Compounds can be analytically characterized byremoving a portion of solution from each well and injecting the contentsinto a separation device such as liquid chromatography or capillaryelectrophoresis instrument coupled to a mass spectrometer. Assuming thatsuch a method would take approximately 5 minutes per analysis, it wouldrequire over a month to analyze the contents of 105 96-well plates,assuming the method was fully automated and operating 24 hours a day.Even larger well-plates containing 384 and 1536 wells are beingintegrated into the screening of new chemical entities imposing evengreater time constraints on the analytical characterization of theselibraries.

Recent technological developments in combinatorial chemistry, molecularbiology, and new microchip chemical devices have created the need fornew types of dispensing devices. Applications in combinatorial chemistryrequire robust sample delivery systems that are chemically inert anddistribute less than microliter amounts of liquid in high-densityformats. The systems need to be highly reproducible and have overallquick dispensing times. Current dispensing technology utilizes serialinjection schemes. The use of serial dispensers will be inherentlylimited due to their slow overall distribution times as the move tohigh-density formats progresses. For example, for combinatorialchemistry applications, to synthesize a library of 1 million discretecompounds, each composed of 4 monomers, a total of 4×106 dispensingsteps would be required. If each dispensing step required 3 seconds(considering dispense time, rinsing, and, location positioning), thetotal time to dispense all of the reagents would be 12×106 seconds, or3333 hours, or 139 days. Thus, for high-density formats, dispensing mustbe conducted in parallel. In order for parallel dispensing to work inhigh-density formats, the dispensing device must be small enough toallow all dispensing units to be simultaneously positioned within acorresponding receiving well. This requires the dispenser to berelatively small. As high density formats reach greater than 10,000wells, dispensing devices will need to be spaced within 100 μm or less.In addition, in order for the dispenser to be practical, the device mustdispense small quantities of liquid (10⁻⁹ to 10⁻¹² L). and only requiresmall volumes to operate.

Piezoelectric dispensing units have also been used for dispensing smallamounts of liquid for microdevices. However, piezoelectric dispenserssuffer from several problems. Currently, the closest spacing ofindividual dispensers is 330 μm in an array of four. Due to the currentpiezoelectric design and fabrication, the number of dispensers that canbe positioned adjacent to one another is limited because of downstreamdevice features. Additionally, sample requirements may be quite higheven though the dispensing volume is small.

Enormous amounts of genetic sequence data are being generated throughnew DNA sequencing methods. This wealth of new information is generatingnew insights into the mechanism of disease processes. In particular, theburgeoning field of genomics has allowed rapid identification of newtargets for drug discovery. Determination of genetic variations betweenindividuals has opened up the possibility of targeting drugs toindividuals based on the individual's particular genetic profile.Testing for cytotoxicity, specificity, and other pharmaceuticalcharacteristics could be carried out in high-throughput assays insteadof expensive animal testing and clinical trials. Detailedcharacterization of a potential drug or lead compound early in the drugdevelopment process thus has the potential for significant savings bothin time and expense.

Development of viable screening methods for these new targets will oftendepend on the availability of rapid separation and analysis techniquesfor analyzing the results of assays. For example, an assay for potentialtoxic metabolites of a candidate drug would need to identify both thecandidate drug and the metabolites of that candidate. An assay forspecificity would need to identify compounds that bind differentially totwo molecular targets such as a viral protease and a mammalian protease.

It would, therefore, be advantageous to provide a method for efficientproteomic screening in order to obtain the pharmacokinetic profile of adrug early in the evaluation process. An understanding of how a newcompound is absorbed in the body and how it is metabolized can enableprediction of the likelihood for an increased therapeutic effect or lackthereof.

Given the enormous number of new compounds that are being generateddaily, an improved system for identifying molecules of potentialtherapeutic value for drug discovery is also critically needed.Accordingly, there is a critical need for high-throughput screening andidentification of compound-target reactions in order to identifypotential drug candidates.

Liquid chromatography (LC) is a well-established analytical method forseparating components of a fluid for subsequent analysis and/oridentification. Traditionally, liquid chromatography utilizes aseparation column, such as a cylindrical tube with dimensions 4.6 mminner diameter by 25 cm length, filled with tightly packed particles of5 μm diameter. More recently, particles of 3 μm diameter are being usedin shorter length columns. The small particle size provides a largesurface area that can be modified with various chemistries creating astationary phase. A liquid eluent is pumped through the LC column at anoptimized flow rate based on the column dimensions and particle size.This liquid eluent is referred to as the mobile phase. A volume ofsample is injected into the mobile phase prior to the LC column. Theanalytes in the sample interact with the stationary phase based on thepartition coefficients for each of the analytes. The partitioncoefficient is defined as the ratio of the time an analyte spendsinteracting with the stationary phase to the time spent interacting withthe mobile phase. The longer an analyte interacts with the stationaryphase, the higher the partition coefficient and the longer the analyteis retained on the LC column. The diffusion rate for an analyte througha mobile phase (mobile-phase mass transfer) also affects the partitioncoefficient. The mobile-phase mass transfer can be rate limiting in theperformance of the separation column when it is greater than 2 μm (Knox,J. H. J. J. Chromatogr. Sci. 18:453-461 (1980)). Increases inchromatographic separation are achieved when using a smaller particlesize as the stationary phase support.

The purpose of the LC column is to separate analytes such that a uniqueresponse for each analyte from a chosen detector can be acquired for aquantitative or qualitative measurement. The ability of a LC column togenerate a separation is determined by the dimensions of the column andthe particle size supporting the stationary phase. A measure of theability of LC columns to separate a given analyte is referred to as thetheoretical plate number N. The retention time of an analyte can beadjusted by varying the mobile phase composition and the partitioncoefficient for an analyte. Experimentation and a fundamentalunderstanding of the partition coefficient for a given analyte determinewhich stationary phase is chosen.

To increase the throughput of LC analyses requires a reduction in thedimensions of the LC column and the stationary phase particledimensions. Reducing the length of the LC column from 25 cm to 5 cm willresult in a factor of 5 decrease in the retention time for an analyte.At the same time, the theoretical plates are reduced 5-fold. To maintainthe theoretical plates of a 25 cm length column packed with 5 μmparticles, a 5 cm column would need to be packed with 1 μm particles.However, the use of such small particles results in many technicalchallenges.

One of these technical challenges is the backpressure resulting frompushing the mobile phase through each of these columns. The backpressureis a measure of the pressure generated in a separation column due topumping a mobile phase at a given flow rate through the LC column. Forexample, the typical backpressure of a 4.6 mm inner diameter by 25 cmlength column packed with 5 μm particles generates a backpressure of 100bar at a flow rate of 1.0 mL/min. A 5 cm column packed with 1 μmparticles generates a back pressure 5 times greater than a 25 cm columnpacked with 5 μm particles. Most commercially available LC pumps arelimited to operating pressures less than 400 bar and thus using an LCcolumn with these small particles is not feasible.

Detection of analytes separated on an LC column has traditionally beenaccomplished by use of spectroscopic detectors. Spectroscopic detectorsrely on a change in refractive index, ultraviolet and/or visible lightabsorption, or fluorescence after excitation with a suitable wavelengthto detect the separated components. Additionally, the effluent from anLC column may be nebulized to generate an aerosol which is sprayed intoa chamber to measure the light scattering properties of the analyteseluting from the column. Alternatively, the separated components may bepassed from the liquid chromatography column into other types ofanalytical instruments for analysis. The volume from the LC column tothe detector is minimized in order to maintain the separation efficiencyand analysis sensitivity. All system volume not directly resulting fromthe separation column is referred to as the dead volume or extra-columnvolume.

The miniaturization of liquid separation techniques to the nano-scaleinvolves small column internal diameters (<100 μm i.d.) and low mobilephase flow rates (<300 nL/min). Currently, techniques such as capillaryzone electrophoresis (CZE), nano-LC, open tubular liquid chromatography(OTLC), and capillary electrochromatography (CEC) offer numerousadvantages over conventional scale high performance liquidchromatography (HPLC). These advantages include higher separationefficiencies, high-speed separations, analysis of low volume samples,and the coupling of 2-dimensional techniques. One challenge to usingminiaturized separation techniques is detection of the small peakvolumes and a limited number of detectors that can accommodate thesesmall volumes. However, coupling of low flow rate liquid separationtechniques to electrospray mass spectrometry results in a combination oftechniques that are well suited as demonstrated in J. N. Alexander IV,et al., Rapid Commun. Mass Spectrom. 12:1187-91 (1998). The process ofelectrospray at flow rates on the order of nanoliters per minute hasbeen referred to as “nanoelectrospray”.

Capillary electrophoresis is a technique that utilizes theelectrophoretic nature of molecules and/or the electroosmotic flow offluids in small capillary tubes to separate components of a fluid.Typically, a fused silica capillary of 100 μm inner diameter or less isfilled with a buffer solution containing an electrolyte. Each end of thecapillary is placed in a separate fluidic reservoir containing a bufferelectrolyte. A potential voltage is placed in one of the bufferreservoirs and a second potential voltage is placed in the other bufferreservoir. Positively and negatively charged species will migrate inopposite directions through the capillary under the influence of theelectric field established by the two potential voltages applied to thebuffer reservoirs. Electroosmotic flow is defined as the fluid flowalong the walls of a capillary due to the migration of charged speciesfrom the buffer solution under the influence of the applied electricfield. Some molecules exist as charged species when in solution and willmigrate through the capillary based on the charge-to-mass ratio of themolecular species. This migration is defined as electrophoreticmobility. The electroosmotic flow and the electrophoretic mobility ofeach component of a fluid determine the overall migration for eachfluidic component. The fluid flow profile resulting from electroosmoticflow is flat due to the reduction in frictional drag along the walls ofthe separation channel. This results in improved separation efficiencycompared to liquid chromatography where the flow profile is parabolicresulting from pressure driven flow.

Capillary electrochromatography is a hybrid technique that utilizes theelectrically driven flow characteristics of electrophoretic separationmethods within capillary columns packed with a solid stationary phasetypical of liquid chromatography. It couples the separation power ofreversed-phase liquid chromatography with the high efficiencies ofcapillary electrophoresis. Higher efficiencies are obtainable forcapillary electrochromatography separations over liquid chromatography,because the flow profile resulting from electroosmotic flow is flat dueto the reduction in frictional drag along the walls of the separationchannel when compared to the parabolic flow profile resulting frompressure driven flows. Furthermore, smaller particle sizes can be usedin capillary electrochromatography than in liquid chromatography,because no backpressure is generated by electroosmotic flow. In contrastto electrophoresis, capillary electrochromatography is capable ofseparating neutral molecules due to analyte partitioning between thestationary and mobile phases of the column particles using a liquidchromatography separation mechanism.

Microchip-based separation devices have been developed for rapidanalysis of large numbers of samples. Compared to other conventionalseparation devices, these microchip-based separation devices have highersample throughput, reduced sample and reagent consumption, and reducedchemical waste. The liquid flow rates for microchip-based separationdevices range from approximately 1-300 nanoliters (nL) per minute formost applications. Examples of microchip-based separation devicesinclude those for capillary electrophoresis (“CE”), capillaryelectrochromatography (“CEC”) and high-performance liquid chromatography(“HPLC”) include Harrison et al., Science 261:859-97 (1993); Jacobson etal., Anal, Chem. 66:1114-18 (1994). Jacobson et al., Anal. Chem.66:2369-73 (1994), Kutter et al., Anal. Chem. 69:5165-71 (1997) and Heet al., Anal. Chem. 70:3790-97 (1998). Such separation devices arecapable of fast analyses and provide improved precision and reliabilitycompared to other conventional analytical instruments.

The work of He et al., Anal. Chem. 70:3790-97 (1998) demonstrates someof the types of structures that can be fabricated in a glass substrate.This work shows that co-located monolithic support structures (or posts)can be etched reproducibly in a glass substrate using reactive ionetching (RIE) techniques. Currently, anisotropic RIE techniques forglass substrates are limited to etching features that are 20 μm or lessin depth. This work shows rectangular 5 μm by 5 μm width by 10 μm indepth posts and stated that deeper structures were difficult to achieve.The posts are also separated by 1.5 μm. The posts supports thestationary phase just as with the particles in LC and CEC columns. Anadvantage to the posts over conventional LC and CEC is that thestationary phase support structures are monolithic with the substrateand therefore, immobile.

He et. al., also describes the importance of maintaining a constantcross-sectional area across the entire length of the separation channel.Large variations in the cross-sectional area can create pressure dropsin pressure driven flow systems. In electrokinetically driven flowsystems, large variations in the cross-sectional area along the lengthof a separation channel can create flow restrictions that result inbubble formation in the separation channel. Since the fluid flowingthrough the separation channel functions as the source and carrier ofthe mobile solvated ions, formation of a bubble in a separation channelwill result in the disruption of the electroosmotic flow.

Electrospray ionization provides for the atmospheric pressure ionizationof a liquid sample. The electrospray process creates highly-chargeddroplets that, under evaporation, create ions representative of thespecies contained in the solution. An ion-sampling orifice of a massspectrometer may be used to sample these gas phase ions for massanalysis. A schematic of an electrospray system 50 is shown in FIG. 1A.An electrospray is produced when a sufficient electrical potentialdifference V_(spray) is applied between a conductive or partlyconductive fluid exiting a capillary 52 and an extracting electrode 54to generate a concentration of electric field lines emanating from thetip or end of a capillary 56. When a positive voltage V_(spray) isapplied to the tip of the capillary relative to an extracting electrode,such as one provided at the ion-sampling orifice of a mass spectrometer,the electric field causes positively-charged ions in the fluid tomigrate to the surface of the fluid at the tip of the capillary. When anegative voltage V_(spray) is applied to the tip of the capillaryrelative to an extracting electrode, such as one provided at theion-sampling orifice to the mass spectrometer, the electric field causesnegatively-charged ions in the fluid to migrate to the surface of thefluid at the tip of the capillary.

When the repulsion force of the solvated ions on the surface of thefluid exceeds the surface tension of the fluid being electrosprayed, avolume of the fluid is pulled into the shape of a cone, known as aTaylor cone 58, which extends from the tip of the capillary 56. A liquidjet 60 extends from the tip of the Taylor cone and becomes unstable andgenerates charged-droplets 62. These small charged droplets are drawntoward the extracting electrode 54. The small droplets arehighly-charged and solvent evaporation from the droplets results in theexcess charge in the droplet residing on the analyte molecules in theelectrosprayed fluid. The charged molecules or ions are drawn throughthe ion-sampling orifice of the mass spectrometer for mass analysis.This phenomenon has been described, for example, by Dole et al., Chem.Phys. 49:2240 (1968) and Yamashita et al., J. Phys. Chem. 88:4451(1984). The potential voltage required to initiate an electrospray isdependent on the surface tension of the solution as described by, forexample, Smith, IEEE Trans. Ind. Appl. 1986, IA-22:527-35 (1986).Typically, the electric field is on the order of approximately 10⁶ V/m.The physical size of the capillary and the fluid surface tensiondetermines the density of electric field lines necessary to initiateelectrospray.

When the repulsion force of the solvated ions is not sufficient toovercome the surface tension of the fluid exiting the tip of thecapillary, large poorly charged droplets are formed as shown in FIG. 1B.Fluid droplets 64 are produced when the electrical potential differenceV_(droplet) applied between a conductive or partly conductive fluidexiting a capillary 52 and an electrode is not sufficient to overcomethe fluid surface tension to form a Taylor cone.

Electrospray Ionization Mass Spectrometry: Fundamentals Instrumentation,and Applications. edited by R. B. Cole, ISBN 0-471-14564-5, John Wiley &Sons, Inc., New York summarizes much of the fundamental studies ofelectrospray. Several mathematical models have been generated to explainthe principals governing electrospray. Equation 1 defines the electricfield E_(c) at the tip of a capillary of radius r_(c) with an appliedvoltage V_(c) at a distance d from a counter electrode held at groundpotential: $\begin{matrix}{E_{c} = \frac{2V_{c}}{r_{c}{\ln \left( {4{d/r_{c}}} \right)}}} & (1)\end{matrix}$

The electric field E_(on) required for the formation of a Taylor coneand liquid jet of a fluid flowing to the tip of this capillary isapproximated as: $\begin{matrix}{E_{on} \approx \left( \frac{2\quad \gamma \quad \cos \quad \theta}{ɛ_{o}r_{c}} \right)^{1/2}} & (2)\end{matrix}$

where γ is the surface tension of the fluid, θ is the half-angle of theTaylor cone and ε₀ is the permittivity of vacuum. Equation 3 is derivedby combining equations 1 and 2 and approximates the onset voltage V_(on)required to initiate an electrospray of a fluid from a capillary:$\begin{matrix}{V_{on} \approx {\left( \frac{r_{c}\gamma \quad \cos \quad \theta}{2\quad ɛ_{0}} \right)^{1/2}{\ln \left( {4{d/r_{c}}} \right)}}} & (3)\end{matrix}$

The graph of FIG. 1C shows curves for onset voltages of 500, 750 and1000 V as related to surface tension of a fluid undergoing electrosprayfrom the tip of a capillary with a given outer diameter. The distance ofthe capillary tip from the counter-electrode was fixed at 2 mm.Combinations of fluid surface tension and capillary diameters that fallbelow the curves will generate a stable electrospray using a given onsetvoltage. As can be seen by examination of equation 3, the required onsetvoltage is more dependent on the capillary radius than the distance fromthe counter-electrode.

It would be desirable to define an electrospray device that could form astable electrospray of all fluids commonly used in CE, CEC, and LC. Thesurface tension of solvents commonly used as the mobile phase for theseseparations range from 100% aqueous (γ=0.073 N/m) to 100% methanol(γ=0.0226 N/m). FIG. 1C indicates that as the surface tension of theelectrospray fluid increases, a higher onset voltage is required toinitiate an electrospray for a fixed capillary diameter. As an example,a capillary with a tip diameter of 14 μm is required to electrospray100% aqueous solutions with an onset voltage of 1000 V. The work of M.S. Wilm et al., Int. J. Mass Spectrom. Ion Processes 136:167-80 (1994),first demonstrates nanoelectrospray from a fused-silica capillary pulledto an outer diameter of 5 μm at a flow rate of 25 nL/min. Specifically,a nanoelectrospray at 25 nL/min was achieved from a 2 μm inner diameterand 5 μm outer diameter pulled fused-silica capillary with 600-700 V ata distance of 1-2 mm from the ion-sampling orifice of an electrosprayequipped mass spectrometer.

Electrospray in front of an ion-sampling orifice of an API massspectrometer produces a quantitative response from the mass spectrometerdetector due to the analyte molecules present in the liquid flowing fromthe capillary. One advantage of electrospray is that the response for ananalyte measured by the mass spectrometer detector is dependent on theconcentration of the analyte in the fluid and independent of the fluidflow rate. The response of an analyte in solution at a givenconcentration would be comparable using electrospray combined with massspectrometry at a flow rate of 100 μL/min compared to a flow rate of 100nL/min. D. C. Gale et al., Rapid Commun. Mass Spectrom. 7:1017 (1993)demonstrate that higher electrospray sensitivity is achieved at lowerflow rates due to increased analyte ionization efficiency.

Attempts have been made to manufacture an electrospray device formicrochip-based separations. Ramsey et al., Anal. Chem. 69:1174-78(1997) describes a microchip-based separations device coupled with anelectrospray mass spectrometer. Previous work from this research groupincluding Jacobson et al., Anal. Chem. 66:1114-18 (1994) and Jacobson etal., Anal. Chem. 66:2369-73 (1994) demonstrate impressive separationsusing on-chip fluorescence detection. This more recent work demonstratesnanoelectrospray at 90 nL/min from the edge of a planar glass microchip.The microchip-based separation channel has dimensions of 10 μm deep, 60μm wide, and 33 mm in length. Electroosmotic flow is used to generatefluid flow at 90 nL/min. Application of 4,800 V to the fluid exiting theseparation channel on the edge of the microchip at a distance of 3-5 mmfrom the ion-sampling orifice of an API mass spectrometer generates anelectrospray. Approximately 12 nL of the sample fluid collects at theedge of the microchip before the formation of a Taylor cone and stablenanoelectrospray from the edge of the microchip. The volume of thismicrochip-based separation channel is 19.8 nL. Nanoelectrospray from theedge of this microchip device after capillary electrophoresis orcapillary electrochromatography separation is rendered impractical sincethis system has a dead-volume approaching 60% of the column (channel)volume. Furthermore, because this device provides a flat surface, and,thus, a relatively small amount of physical asperity for the formationof the electrospray, the device requires an impractically high voltageto overcome the fluid surface tension to initiate an electrospray.

Xue, Q. et al., Anal. Chem. 69:426-30 (1997) also describes a stablenanoelectrospray from the edge of a planar glass microchip with a closedchannel 25 μm deep, 60 μm wilde, and 35-50 mm in length. An electrosprayis formed by applying 4,200 V to the fluid exiting the separationchannel on the edge of the microchip at a distance of 3-8 mm from theion-sampling orifice of an API mass spectrometer. A syringe pump isutilized to deliver the sample fluid to the glass microchip at a flowrate of 100 to 200 nL/min. The edge of the glass microchip is treatedwith a hydrophobic coating to alleviate some of the difficultiesassociated with nanoelectrospray from a flat surface that slightlyimproves the stability of the nanoelectrospray. Nevertheless, the volumeof the Taylor cone on the edge of the microchip is too large relative tothe volume of the separation channel, making this method of electrospraydirectly from the edge of a microchip impracticable when combined with achromatographic separation device.

T. D. Lee et. al., 1997 International Conference on Solid-State Sensorsand Actuators Chicago, pp. 927-30 (Jun. 16-19, 1997) describes amulti-step process to generate a nozzle on the edge of a siliconmicrochip 1-3 μm in diameter or width and 40 μm in length and applying4,000 V to the entire microchip at a distance of 0.25-0.4 mm from theion-sampling orifice of an API mass spectrometer. Because a relativelyhigh voltage is required to form an electrospray with the nozzlepositioned in very close proximity to the mass spectrometer ion-samplingorifice, this device produces an inefficient electrospray that does notallow for sufficient droplet evaporation before the ions enter theorifice. The extension of the nozzle from the edge of the microchip alsoexposes the nozzle to accidental breakage. More recently, T. D. Lee et.al., in 1999 Twelfth IEEE International Micro Electro Mechanical SystemsConference (Jan. 17-21, 1999), presented this same concept where theelectrospray component was fabricated to extend 2.5 mm beyond the edgeof the microchip to overcome this phenomenon of poor electric fieldcontrol within the proximity of a surface.

In all of the above-described devices, generating an electrospray fromthe edge of a microchip is a poorly controlled process. These devices donot define a nozzle and an electric field around the nozzle that isrequired to produce a stable and highly reproducible electrospray. Inanother embodiment, small segments of fused-silica capillaries areseparately and individually attached to the chip's edge. This process isinherently cost-inefficient and unreliable, imposes space constraints inchip design, and is therefore unsuitable for manufacturing.

Thus, it is also desirable to provide an electrospray device withcontrollable spraying and a method for producing such a device that iseasily reproducible and manufacturable in high volumes.

U.S. Pat. No. 5,501,893 to Laermer et. al., reports a method ofanisotropic plasma etching of silicon (Bosch process) that provides amethod of producing deep vertical structures that is easily reproducibleand controllable. This method of anisotropic plasma etching of siliconincorporates a two step process. Step one is an anisotropic etch stepusing a reactive ion etching (RIE) gas plasma of sulfur hexafluoride(SF₆). Step two is a passivation step that deposits a polymer on thevertical surfaces of the silicon substrate. This polymerizing stepprovides an etch stop on the vertical surface that was exposed in stepone. This two step cycle of etch and passivation is repeated until thedepth of the desired structure is achieved. This method of anisotropicplasma etching provides etch rates over 3 μm/min of silicon depending onthe size of the feature being etched. The process also providesselectivity to etching silicon versus silicon dioxide or resist ofgreater than 100:1 which is important when deep silicon structures aredesired. Laermer et. al., in 1999 Twelfth IEEE International MicroElectro Mechanical Systems Conference (Jan. 17-21, 1999), reportedimprovements to the Bosch process. These improvements include siliconetch rates approaching 10 μm/min, selectivity exceeding 300:1 to silicondioxide masks, and more uniform etch rates for features that vary insize.

The electrical properties of silicon and silicon-based materials arewell characterized. The use of silicon dioxide and silicon nitridelayers grown or deposited on the surfaces of a silicon substrate arewell known to provide electrical insulating properties. Silicon dioxidelayers may be grown thermally in an oven to a desired thickness. Siliconnitride can be deposited using low pressure chemical vapor deposition(LPCVD). Metals may be further vapor deposited on these surfaces toprovide for application of a potential voltage on the surface of thedevice. Both silicon dioxide and silicon nitride function as electricalinsulators allowing the application of a potential voltage to thesubstrate that is different than that applied to the surface of thedevice. An important feature of a silicon nitride layer is that itprovides a moisture barrier between the silicon substrate, silicondioxide and any fluid sample that comes in contact with the device.Silicon nitride prevents water and ions from diffusing through thesilicon dioxide layer to the silicon substrate which may cause anelectrical breakdown between the fluid and the silicon substrate.Additional layers of silicon dioxide, metals and other materials mayfurther be deposited on the silicon nitride layer to provide chemicalfunctionality to silicon-based devices.

The present invention is directed to overcoming the deficiencies inprior electrospray systems.

SUMMARY OF THE INVENTION

The present invention relates to an electrospray device which comprisesa substrate having an injection surface and an ejection surface opposingthe injection surface with the substrate being an integral monolith. Anentrance orifice is positioned on the injection surface, while an exitorifice is on the ejection surface. A channel extends between theentrance orifice and the exit orifice. A recess surrounds the exitorifice and is positioned between the injection surface and the ejectionsurface. The electrospray device has voltage application systemconsisting essentially of a first electrode attached to the substrate toimpart a first potential to the substrate and a second electrode toimpart a second potential, where the first and the second electrodes arepositioned to define an electric field surrounding the exit orifice.This device can be used in conjunction with systems for processingdroplet/sprays, methods of generating an electrospray, a method of massspectrometeric analysis, and a method of liquid chromatographicanalysis.

Another aspect of the present invention is directed to an electrospraydevice which includes a capillary tube having a passage for conductingfluids through the capillary tube and connecting an entrance orifice andan exit orifice, a first electrode circumscribing the capillary tubeproximate the exit orifice, and a second electrode to impart a secondpotential. The first and the second electrodes are positioned to definean electric field surrounding the exit orifice.

Another aspect of the present invention relates to a method of producingan electrospray device which includes providing a substrate havingopposed first and second surfaces, each coated with a photoresist. Thephotoresist on the first surface is exposed to an optical image to forma pattern is the form of a spot on the first surface. The photoresist onthe first surface where the pattern is removed to form a hole in thephotoresist. Material is removed from the substrate coincident with thehole in the photoresist on the first surface to form a channel extendingthrough the photoresist on the first surface and through the substrateup to the photoresist on the second surface. The photoresist on thesecond surface is exposed to an image to form an annular patterncircumscribing an extension of the channel through the photoresist onthe second surface. The photoresist on the second surface having theannular pattern is then removed, and, next, the material from thesubstrate coincident with the removed annular pattern in thephototresist on the second surface is removed to form an annular recessextending partially into the substrate. All coatings from the first andsecond surfaces of the substrate are removed to form the electrospraydevice.

Another aspect of the present invention relates to a method of producingan electrospray device. This method includes providing a substratehaving opposed first and second surfaces, each coated with aphotoresist. The photoresist is exposed on the first surface to an imageto form a pattern in the form of at least 3 substantially aligned spotson the first surface. The photoresist on the first surface is removedwhere the pattern is to form 3 holes in the photoresist corresponding towhere the spots in the photoresist were. Material from the substratecoincident with the removed pattern in the photoresist on the firstsurface is then removed to form a central channel aligned with andbetween two outer channels. The channels extend through the photoresiston the first surface and into the substrate. The central channel has adiameter which is less than that of the outer channels such that thecentral channel extends farther from the second surface of the substratethan the outer channels which extend up to the photoresist on the thesecond surface. The photoresist on the second surface is exposed to animage which forms an annular pattern circumscribing a spot, where thespot is coincident with an extension of the central channel through thephotoresist on the second surface and a portion of the substrate. Thephotoresist on the second surface is removed where the annular patterncircumscribing the spot is. Material from the substrate coincident withthe removed pattern in the photoresist on the second surface is thenremoved. This forms an annular recess extending partially into thesubstrate and circumscribing the central channel which extends throughthe substrate and the photoresist on the first and second surfaces. Allcoatings from the first and second surfaces of the substrate are thenremoved. All surfaces of the substrate are then coated with aninsulating material to form the electrospray device.

Another aspect of the present invention relates to a method of forming aliquid separation device. This method involves providing a substratehaving opposed first and second surfaces, each coated with aphotoresist. The photoresist is exposed on the first surface to an imageto form a pattern in the form of a plurality of spots on the firstsurface. The photoresist on the first surface where the pattern is isremoved to form a plurality of holes in the photoresist corresponding towhere the spots in the photoresist were. Material from the substratecoincident with where the pattern in the photoresist on the firstsurface has been removed is then removed. This forms a large reservoirproximate a first end of the substrate and a plurality of smaller holescloser to a second opposite end of the substrate than the reservoir. Thereservoir and holes extend through the photoresist on the first surfaceand partially into the substrate. The smaller holes and the surfaces ofthe reservoir are filled with a coating, and a further photoresist layeris applied over the coating on the surfaces of the reservoir, the filledholes, and the photoresist on the first surface. The further photoresistis exposed to an image to form a pattern in the form of spots, with onespot coincident with what was the reservoir and the other spot beingcloser to the second end of the substrate than the filled holes. Thefurther photoresist is removed where the pattern is to form holescorresponding to where the spots in the photoresist were. Material isremoved from the substrate coincident with where the pattern in thefurther photoresist has been removed to form a pair of channels. A firstchannel extends through what was the reservoir up to the photoresist onthe second surface. A second channel extends through the substrate up tothe photoresist on the second surface at a location closer to the secondend of the substrate than the filled holes. All coatings from the firstand second surfaces of the substrate are removed, and all surfaces ofthe substrate are coated with an insulating material to form the liquidseparation device.

Another aspect of the present invention relates to a system forprocessing droplets/sprays of fluid which includes an electrospraydevice. The electrospray device contains a substrate having an injectionsurface and an ejection surface opposing the injection surface. Thesubstrate comprises an entrance orifice on the injection surface, anexit orifice on the ejection surface, a channel extending between theentrance orifice and the exit orifice, and a recess extending into theejection surface and surrounding the exit orifice. The system furtherincludes a device to provide fluid to the electrospray device whichincludes a fluid passage, a fluid reservoir in fluid communication withthe fluid passage, a fluid inlet to direct fluid entering the deviceinto the fluid reservoir, and a fluid outlet to direct fluid from thefluid passage to the entrance orifice of the electrospray device. Thecross-sectional area of the entrance orifice of the electrospray deviceis equal to or less than the cross-sectional area of the fluid passage.

The present invention achieves a significant advantage in terms ofhigh-throughput distribution and apportionment of massively parallelchannels of discrete chemical entities in a well-controlled,reproducible method. An array of dispensing nozzles is disclosed forapplication in inkjet printing. When combined with a miniaturized liquidchromatography system and method, the present invention achieves asignificant advantage in comparison to a conventional system.

The present invention insulates a fluid introduced to the electrospraydevice from the silicon substrate of the device. This insulation is inthe form of silicon dioxide and silicon nitride layers contained on thesurfaces of the electrospray device. These insulating layers allow forindependent application of a voltage to the fluid introduced to theelectrospray device and the voltage applied to the substrate. Theindependent voltage application to the fluid and substrate allow forcontrol of the electric field around the exit orifice of the nozzle onthe ejection surface of the electrospray device independent of the needfor any additional electrodes or voltages. This, combined with thedimensions of the nozzle formed from the ejection surface of theelectrospray device and the fluid surface tension, determine theelectric field and voltages required for the formation of droplets or anelectrospray from this invention.

The electrospray device of the present invention can be integrated withmicrochip-based devices having atmospheric pressure ionization massspectrometry (API-MS) instruments. By generating an electric field atthe tip of a nozzle, which exists in a planar or near planar geometrywith the ejection surface of a substrate, fluid droplets and anelectrospray exiting the nozzle on the ejection surface are efficientlygenerated. When a nozzle exists in this co-planar or near planargeometry, the electric field lines emanating from the tip of the nozzlewill not be enhanced if the electric field around the nozzle is notdefined and controlled.

Control of the electric field at the tip of a nozzle formed from asubstrate for the efficient formation of droplets and electrospray froma microchip is an important aspect of the present invention. This wasdetermined using a fused-silica capillary 52 pulled to an outer diameterof approximately 20 μm and inserted through a ring electrode 70 with a 1mm diameter as shown in FIG. 2. FIG. 2A shows a plan view of thecapillary/ring electrode experiment. FIG. 2B shows a cross-sectionalview of the capillary/ring electrode experiment. The capillary tip 56 isinserted up to 5 mm through the ring electrode 70 in front of anion-sampling orifice 54 of a mass spectrometer equipped with anelectrospray ion source. A voltage of 700 V is applied to an aqueousfluid V_(fluid) flowing to the capillary tip at a flow rate of 50nL/min. The ring electrode 70 is mounted on an XYZ stage to allow thering electrode to be moved slowly forward to the point at which thecapillary tip 56 is co-planar with the ring electrode 70 as shown inFIGS. 2C and 2D. The voltage applied to the ring electrode V_(ring) isvariable. The voltage applied to the ion-sampling orifice 54 is 80 V.When the fluid voltage and the ring electrode voltage are maintained at700 V in the co-planar geometry, the electrospray is disrupted and nolonger forms an electrospray. Depending on the V_(fluid)/V_(ring) ratiofor a fixed distance from a counter electrode 54, fluidic droplets canbe controllably dispensed from the capillary tip as shown in FIG. 2C. Inthis case, minimally-charged, larger droplets are formed with thedroplet diameter dependent on the electric field established by theV_(fluid)/V_(ring) ratio, fluid surface tension, fluid conductivity,capillary tip diameter and distance from a counter electrode.Application of a voltage of 0 V to the ring electrode 70 results in theformation of a stable electrospray once again as shown in FIG. 2D. FIG.2D shows a Taylor cone 58, liquid jet 60 and plume of highly-chargeddroplets 62.

The response of the analyte measured by the mass spectrometer detectorincreases beyond that of a capillary with no ring electrode present uponincreasing the ring electrode voltage to 350 V. A V_(fluid)/V_(ring)ratio of less than approximately two for a fixed distance from a counterelectrode reduces the electric field at the capillary tip to the pointwhere a stable electrospray is no longer sustainable and larger dropletformation is observed. These results indicate that an important featureof any integrated monolithic device designed for droplet formation orelectrospray is control of the electric field around the orifice of anozzle in a co-planar or near planar geometry.

The present invention provides a microchip-based electrospray device forproducing reproducible, controllable and robust nanoelectrospray of aliquid sample. The electrospray device is designed to enhance theelectric field emanating from a nozzle etched from a surface of amonolithic silicon substrate. This is accomplished by providinginsulating layers of silicon dioxide and silicon nitride, for example,for independent application of a potential voltage to a fluid exiting atthe tip of the nozzle from a potential voltage applied to the substratesufficient to cause an electrospray of the fluid. The enhanced electricfield combined with the physical asperity of the nozzle allow for theformation of an electrospray of fluids at flow rates as low as a fewnanoliters per minute. The large electric field, on the order of 10⁶ V/mor greater and generated by the potential difference between the fluid,and the substrate is thus applied directly to the fluidic cone ratherthan uniformly distributed in space.

To generate an electrospray, fluid may be delivered to thethrough-substrate channel of the electrospray device by, for example, acapillary, micropipette or microchip. The fluid is subjected to apotential voltage V_(fluid) via an electrode provided on the injectionsurface and isolated from the surrounding surface region and thesubstrate. A potential voltage V_(substrate) may also be applied to thesilicon substrate the magnitude of which is preferably adjustable foroptimization of the electrospray characteristics. The fluid flowsthrough the channel and exits from the nozzle in the form of a Taylorcone, liquid jet, and very fine, highly charged fluidic droplets. It isthe relative electric potential difference between the fluid andsubstrate voltages that affect the electric field. This inventionprovides a method of controlling the electric field at the tip of anozzle to achieve the desired electric field for the application.

The method of fabricating an electrospray device in accordance with thepresent invention is also advantageous. After injection side processingis completed, the through-substrate channel is etched to a final depth,the photoresist is removed, and the substrate is subjected to anelevated temperature in an oxidizing ambient environment to grow 1-4 μmof silicon dioxide on the walls of the through-substrate channel. Thislayer of silicon dioxide on the walls of the through-substrate channelprovides an etch-stop during further processing of the substrate todefine the recessed annular region. The recessed annular region may bepatterned and etched from either the injection or ejection side of thesubstrate when the through-substrate channel is etched through theentire silicon substrate to the silicon dioxide etch stop on theejection side of the substrate. If the through-substrate channel is notetched completely through the substrate, the recessed annular region isetched from the ejection side of the substrate. The recessed annularregion may be patterned and etched to form the silicon dioxide nozzlefor injection side processing or for ejection side processing.

This method does not require high alignment accuracy of features fromthe injection and ejection side processing to define the nozzle wallthickness thus simplifying the method. This method allows nozzles ofdecreasing size to be reproducibly manufactured and does not require thethrough-substrate channel to be etched completely through the substrate.The silicon dioxide layer that is grown on the walls of thethrough-substrate channel determines the wall thickness of the nozzlesusing this method. The desired nozzle size and use of the electrospraydevice determines which method is preferred. This fabrication sequenceconfers superior mechanical stability to the fabricated electrospraydevice by etching the features of the electrospray device from amonocrystalline silicon substrate without any need for assembly.Further, use of a visible alignment mark as described in the fabricationsequence of this device allows for alignment of injection side andejection side features to better than 1 μm. This allows for overallnozzle dimensions that are smaller than previously achieved that useprior disclosed alignment schemes using infrared light. Control of thelateral extent and shape of the recessed annular region provides theability to modify and control the electric field between theelectrospray device and an extracting electrode.

This fully integrated monolithic electrospray device may be coupled witha miniaturized monolithic chromatography or other liquid sample handingdevices. In particular, the electrospray device used as a means ofproducing a fluidic cone for spectroscopic detection including laserinduced fluorescence, ultraviolet absorption, and evaporative lightscattering and mass spectrometry detection. An excitation sourceprovides a light beam. A detector detects the emission or absorbance orlight scattering properties of the analytes in the fluidic Taylor cone.

The microchip-based electrospray device of the present inventionprovides minimal extra-column dispersion as a result of a reduction inthe extra-column volume and provides efficient, reproducible, reliableand rugged formation of an electrospray. This electrospray device isperfectly suited as a means of electrospray of fluids frommicrochip-based separation devices. The design of this electrospraydevice is also robust such that the device can be readily mass-producedin a cost-effective, high-yielding process.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A shows a schematic of an electrospray system emitting smallcharged droplets.

FIG. 1B shows a schematic of an electrospray system emitting poorlycharged droplets.

FIG. 1C shows a graph plotting the surface tension of a solution versusthe capillary tip diameter for onset voltages of 500, 750, 1000, and1500 volts and a 2 mm distance between the capillary tip andcounterelectrode of an electrospray system.

FIGS. 2A to D show an electrospray system and the effect of the positionof the capillary tip relative to the ring electrode on the dropletdiameter of the spray.

FIGS. 3A to C show, respectively, a perspective view, a plan view, and across-sectional view of an electrospray device in accordance with thepresent invention. FIG. 3C is a cross-sectional view taken along line3C—3C of FIG. 3B. FIG. 3D shows a cross-sectional view of an alternativeembodiment of an electrospray device in accordance with the presentinvention. FIGS. 3E and 3F show the use of the electrospray device ofthe present invention to generate a fine spray and minimally chargeddroplets. FIG. 3G shows the use of the electrospray device of thepresent invention in conjunction with a minaturized monolithicchromatography or other liquid sample handling device. FIG. 3H is across-sectional view showing the electrospray device of the presentinvention coupled with a fluidic probe. FIG. 3I is a perspective view ofthe injection side of the electrospray device of FIG. 3H.

FIG. 4A is a photograph showing an electrospray device in accordancewith the present invention. FIG. 4B is a perspective view of anelectrospray device in accordance with the present invention. FIG. 4Cshows a perspective view of an electrospray device in accordance withthe present invention generating an electrospray. FIG. 4D is a massspectrum of a Resperine solution sprayed from the electrospray device ofthe present invention. FIG. 4E is a mass spectrum of 1 nM of CytochromeC solution sprayed from the electrospray device of the presentinvention. FIG. 4F is a mass spectrum of 0.1 nM of Cytochrome C solutionsprayed from the electrospray device of the present invention.

FIGS. 5A to 5B show a perspective view and a cross-sectional view,respectively, of a multiple array of electrospray devices in accordancewith the present invention. FIG. 5B is taken along line 5B—5B of FIG.5A.

FIGS. 6A to 6B show perspective views of alternative embodiments ofmicrochip-based liquid chromatography devices in accordance with thepresent invention. FIG. 6C is a cross-sectional view of themicrochip-based liquid chromatography device of FIG. 6B taken along line6C—6C.

FIGS. 7A to 7F show different separation post spacings.

FIGS. 8A to 8B show plan views of a computer-aided layout of a channelcontaining spaced posts for use in a liquid chromatography device inaccordance with the present invention.

FIGS. 9A-9E show one embodiment of a fabrication sequence for theinjection side of an electrospray device;

FIGS. 10A-10F show another embodiment of a fabrication sequence for theejection side of an electrospray device;

FIGS. 11A-11D show another embodiment of a fabrication sequence of theinjection side of an electrospray device wherein a separatethrough-substrate alignment channel is incorporated into the devicelayout;

FIGS. 12A-12E show another embodiment of a fabrication sequence of theejection side of an electrospray device wherein a separatethrough-substrate alignment channel is incorporated into the devicelayout;

FIG. 13 shows an electrospray device with a sacrificial silicon dioxidelayer;

FIG. 14 shows an electrospray device with a silicon dioxide and asilicon nitride layer;

FIG. 15 shows an electrospray device with a silicon dioxide, a siliconnitride layer, and a conductive metal electrode on the edge of thesilicon substrate;

FIGS. 16A-16I show an embodiment of a chromatography side fabricationsequence of an integrated liquid chromatography-electrospray device;

FIGS. 17A-17D show another embodiment of an electrospray sidefabrication sequence of an integrated liquid chromatography-electrospraydevice.

DETAILED DESCRIPTION OF THE INVENTION

FIGS. 3A, 3B and 3C show, respectively, a perspective view, a plan viewand a cross-sectional view of an electrospray device 100 of the presentinvention. The electrospray apparatus of the present invention generallycomprises a silicon substrate, microchip or wafer 102 defining a throughsubstrate channel 104 between an entrance orifice 106 on an injectionsurface 108 and a nozzle 110 on an ejection surface 112. The channel mayhave any suitable cross-sectional shape such as circular or rectangular.The nozzle 110 has an inner and an outer diameter and is defined by arecessed region 114. The region 114 is recessed from the ejectionsurface 112, extends outwardly from the nozzle 110 and may be annular.The tip of the nozzle 110 does not extend beyond and is preferablycoplanar or level with the ejection surface 112 to thereby protect thenozzle 110 from accidental breakage.

Preferably, the injection surface 108 is opposite the ejection surface112. However, the injection surface may be adjacent to the ejectionsurface such that the channel extending between the entrance orifice andthe nozzle makes a turn within the device. In such a configuration, theelectrospray device would comprise two substrates bonded together. Thefirst substrate may define a through-substrate channel extending betweena bonding surface and the ejection surface, opposite the bondingsurface. The first substrate may further define an open channel recessedfrom the bonding surface extending from an orifice of thethrough-substrate channel and the injection surface such that thebonding surface of the second substrate encloses the open channel uponbonding of the first and second substrates. Alternatively, the secondsubstrate may define an open channel recessed from the bonding surfacesuch that the bonding surface of the first substrate encloses the openchannel upon bonding of the first and second substrates. In yet anothervariation, the first substrate may further define a secondthrough-substrate channel while the open channel extends between the twothrough-substrate channels. Thus, the injection surface is the samesurface as the ejection surface.

The electrospray device 100 further comprises a layer of silicon dioxide118 and a layer of silicon nitride 120 over the injection 108, ejection112, and through-substrate channel 104 surfaces of the substrate 102. Anelectrode 122 is in contact with the substrate 102 on the edge 124 ofthe silicon substrate. The silicon dioxide 118 and silicon nitride 120formed on the walls of the channel 104 electrically isolates a fluidtherein from the silicon substrate 102 and, thus, allows for theindependent application and sustenance of different electricalpotentials to the fluid in the channel 104 and to the silicon substrate102. Additional layers of silicon dioxide or other materials may befurther deposited to provide for any required chemical functionality tothe surface of the device. The ability to independently vary the fluidand substrate potentials allows the optimization of the electrospraythrough modification of the electric field, as described below.

Alternatively as shown in FIG. 3D, the substrate 102 can be inelectrical contact with the fluid in the through-substrate channel whenappropriate for a given application. This is accomplished by selectivedeposition of silicon dioxide on the injection and ejection surfaces ofthe substrate and the through-substrate channel, followed by a selectivedeposition of silicon nitride 120 on the ejection surface. A region ofthe ejection surface 112 exterior to the nozzle 110 may provide asurface on which a conductive electrode 122 may be formed to modify theelectric field between the ejection surface 112, including the nozzletip 110, and the extracting electrode 54. In this case, the substratepotential voltage controls the electric field around the nozzle, thecontrolling electrodes 122 on the ejection surface 112 of the substrate102, and the distance from the counter electrode 54.

As shown in FIG. 3E, to generate an electrospray, fluid may be deliveredto the through-substrate channel 104 of the electrospray device 100 by,for example, a capillary 52, micropipette or microchip. The fluid issubjected to a potential voltage V_(fluid) via a wire positioned in thecapillary 52 or in the channel 104 or via an electrode provided on theinjection surface 108 and isolated from the surrounding surface regionand the substrate 102. A potential voltage V_(substrate) may also beapplied to the electrode 122 on the edge 124 of the silicon substrate102 the magnitude of which is preferably adjustable for optimization ofthe electrospray characteristics. The fluid flows through the channel104 and exits from the nozzle 110 in the form of a Taylor cone 58,liquid jet 60, and very fine, highly charged fluidic droplets 62. Theelectrode 54 may be held at a potential voltage V_(extract) such thatthe highly-charged fluidic droplets are attracted toward the extractingelectrode 54 under the influence of an electric field.

In one embodiment, the nozzle 110 may be placed up to 10 mm from theion-sampling orifice of an API mass spectrometer that may function asthe extracting electrode 54. A potential voltage V_(fluid) ranging fromapproximately 500-1000 V, such as 700 V, is applied to the fluid. Apotential voltage of the substrate V_(substrate) of less than half ofthe fluid potential voltage V_(fluid), or 0-350 V, is applied to thesubstrate to enhance the electric field strength at the tip of thenozzle 110. The extracting electrode 54 may be held at or near groundpotential V_(extract) (0 V). Thus, a nanoelectrospray of a fluidintroduced to the electrospray device 100 is attracted toward theextracting electrode 54.

The nozzle 110 provides the physical asperity to promote the formationof a Taylor cone and efficient electrospray of a fluid. The nozzle 110also forms a continuation of and serves as an exit orifice of thethrough-substrate channel 104. The recessed region 114 serves tophysically isolate the nozzle 110 from the ejection surface 112. Thepresent invention allows the optimization of the electric field linesemanating from the fluid exiting the nozzle 110 through independentcontrol of the potential voltage V_(fluid) of the fluid and thepotential voltage V_(substrate) of the substrate.

Dimensions of the electrospray device 100 can be determined according tovarious factors such as the specific application, the layout design aswell as the upstream and/or downstream device to which the electrospraydevice 100 is interfaced or integrated. Further, the dimensions of thechannel and nozzle may be optimized for the desired flow rate of thefluid sample. The use of reactive-ion etching techniques allows for thereproducible and cost effective production of small diameter nozzles,for example, a 2 μm inner diameter and 5 μm outer diameter.

In one currently preferred embodiment, the silicon substrate 102 of theelectrospray device 100 is approximately 250-300 μm in thickness and thecross-sectional area of the through-substrate channel 104 is less thanapproximately 2,500 μm². Where the channel 104 has a circularcross-sectional shape, the channel 104 and the nozzle 110 have an innerdiameter of up to 50 μm, more preferably up to 30 μm; the nozzle 110 hasan outer diameter of up to 60 μm, more preferably up to 40 μm: andnozzle 110 has a height of (and the recessed portion 114 has a depth of)up to 100 μm. The recessed portion 114 preferably extends up to 300 μmoutwardly from the nozzle 110. The silicon dioxide layer 118 has athickness of approximately 1-4 μm, preferably 1-3 μm. The siliconnitride layer 120 has a thickness of approximately less than 2 μm.

Furthermore, the electrospray device may be operated to produce larger,minimally-charged droplets 126 as shown in FIG. 3F. This is accomplishedby decreasing the electric field at the nozzle exit to a value less thanthat required to generate an electrospray of a given fluid. Adjustingthe ratio of the potential voltage V_(fluid) of the fluid and thepotential voltage V_(substrate) of the substrate controls the electricfield. A V_(fluid)/V_(substrate) ratio approximately less than 2 isrequired for droplet formation. The droplet diameter in this mode ofoperation is dependent on the nozzle diameter, electric field strength,and fluid surface tension. This mode of operation is ideally suited forconveyance and/or apportionment of a multiplicity of discrete amounts offluids, and may find use in such devices as ink jet printers andequipment and instruments requiring controlled distribution of fluids.

This fully integrated monolithic electrospray device may be coupled witha miniaturized monolithic chromatography or other liquid sample handingdevices. FIG. 3G shows this electrospray device used as a means ofproducing a fluidic cone for spectroscopic detection including laserinduced fluorescence, ultraviolet absorption, and evaporative lightscattering and mass spectrometry detection. An excitation source 128provides a light beam 130. A detector 132 detects the emission orabsorbance or light scattering properties of the analytes in the fluidicTaylor cone 58, liquid jet 60, or highly-charged droplets 62.

FIG. 3H shows the use of the electrospray device of the presentinvention interfaced with a liquid sample handling device showing ameans of sealing the liquid handling device to the injection side of thepresent invention. The figure shows an O-ring seal 107 between theliquid sample handling device 52 and the electrospray device 110. FIG.3I shows an array of electrospray devices 106 fabricated on a monolithicsubstrate 100 and interfacing to a liquid sample handling device 52.More than one liquid sample handling device could be interfaced with anarray of electrospray devices. Only one is shown for clarity.

FIG. 4A shows a perspective view picture (approximately 80 timesmagnified) of an electrospray device 100 consisting of a nozzle etchedin a silicon substrate. FIG. 4B is a perspective view on an electrospraydevice in accordance with the present invention. Nozzle 110 has a 20 μmouter diameter and 15 μm inner diameter (through-substrate channel) witha height of 70 μm. The nozzle walls are 2.5 μm in thickness. Therecessed annular region 114 has a radius of 300 μm. The substrate 102has a thickness of 254 μm. FIG. 4C shows a perspective view picture ofan electrospray device generating an electrospray. In this figure, a 50%water:50% methanol solution containing 500 ng/mL of reserpine is beingintroduced to the injection side 108 of the through-substrate channel104 as shown in FIG. 3G. The fluid flow is controlled using a syringepump set at a flow rate of 100 nL/min. A fluid voltage of 700 V isapplied to a stainless steel capillary 52 (not shown) with the substrateheld at zero V. The counter electrode 54 (not shown) is an ion-samplingorifice of a Micromass LCT time-of-flight mass spectrometer held at 80V. The nozzle is approximately 5 mm from the ion-sampling orifice of themass spectrometer. Labeled in FIG. 4C is a real Taylor cone emanatingfrom a nozzle. a liquid jet, a plume of highly-charged droplets and arecessed annular region.

FIG. 4D shows the electrospray mass spectrum acquired from theelectrospray shown in FIG. 4C for the Reserpine solution. Reserpine hasa molecular weight of 608 Da. Electrospray in positive ion mode resultsin the protonation of the molecular molecule resulting in an ion at m/z609. A region of the m/z range from 608 to 613 is inserted to show theseparation of the isotopes of reserpine. FIG. 4E shows the electrospraymass spectrum acquired from the electrospray of a 1 nM (1 femtomole permicroliter) solution of Cytochrome C in 100% water. The solution flowrate is 100 nL/min with a fluid voltage of 1350 V and a substratevoltage of zero V. The mass spectrum shows the multiple-chargedistribution characteristic of large biomolecules from electrosprayionization (peaks are labeled with the respective charge state). FIG. 4Fshows the electrospray mass spectrum acquired from the electrospray of a0.1 nM (100 attomole per microliter) solution of Cytochrome C in 100%water at a flow rate of 100 nL/min.

The electrospray device of the present invention generally comprises asilicon substrate material defining a channel between an entranceorifice on an injection surface and a nozzle on an ejection surface (themajor surface) such that the electrospray generated by the device isgenerally perpendicular to the ejection surface. The nozzle has an innerand an outer diameter and is defined by an annular portion recessed fromthe ejection surface. The annular recess extends radially from the outerdiameter. The tip of the nozzle is co-planar or level with and does notextend beyond the ejection surface. Thus, the nozzle is protectedagainst accidental breakage. The nozzle, the channel, and the recessedportion are etched from the silicon substrate by reactive-ion etchingand other standard semiconductor processing techniques.

All surfaces of the silicon substrate preferably have insulating layersto electrically isolate the liquid sample from the substrate and theejection and injection surfaces from each other such that differentpotential voltages may be individually applied to each surface and theliquid sample. The insulating layer generally consists of a silicondioxide layer combined with a silicon nitride layer. The silicon nitridelayer provides a moisture barrier against water and ions frompenetrating through to the substrate causing electrical breakdownbetween a fluid moving in the channel and the substrate. Theelectrospray apparatus further comprises at least one controllingelectrode electrically contacting the substrate for the application ofan electric potential to the substrate.

In another embodiment, all surfaces of the silicon substrate haveinsulating layers thereon to electrically isolate all surfaces of thesubstrate from each other such that different potential voltages may beindividually applied to each surface and the liquid. The insulatinglayer is selectively removed from the tip of the nozzle therefore,making an electrical contact between the tip of the nozzle and thesubstrate. Fluid exiting the nozzle will be at the potential voltageapplied to the substrate. A layer of conductive metal may be selectivelydeposited on the ejection surface of the substrate to provide forenhancement of the electric field at the tip of the nozzle.Alternatively, this electrode may be removed from the substratealtogether and reside in close proximity to the ejection surface of thesubstrate to enhance the electric field emanating from the tip of thenozzle when held at an appropriate voltage. One advantage to this designis that the insulating layer on the surface of the silicon substrate nolonger determines the maximum difference in the voltage applied to thefluid relative to the substrate used to enhance the electric field atthe tip of the nozzle. This will allow for higher potential voltages tobe applied to the fluid and, therefore, provide greater flexibility inthe optimization of the electrospray.

Preferably, the nozzle, channel and recess are etched from the siliconsubstrate by reactive-ion etching and other standard semiconductorprocessing techniques. The injection-side features, through-substratefluid channel, ejection-side features, and controlling electrodes areformed monolithically from a monocrystalline silicon substrate—i.e.,they are formed during the course of and as a result of a fabricationsequence that requires no manipulation or assembly of separatecomponents.

Because the electrospray device is manufactured using reactive-ionetching and other standard semiconductor processing techniques, thedimensions of such a device can be very small, for example, as small as2 μm inner diameter and 5 μm outer diameter. Thus, a through-substratefluid channel having, for example, 5 μm inner diameter and a substratethickness of 250 μm only has a volume of 4.9 pL (picoliters). Themicrometer-scale dimensions of the electrospray device minimize the deadvolume and thereby increase efficiency and analysis sensitivity whencombined with a separation device.

The electrospray device of the present invention provides for theefficient and effective formation of an electrospray. By providing anelectrospray surface from which the fluid is ejected with dimensions onthe order of micrometers, the electrospray device limits the voltagerequired to generate a Taylor cone as the voltage is dependent upon thenozzle diameter, the surface tension of the fluid, and the distance ofthe nozzle from an extracting electrode. The nozzle of the electrospraydevice provides the physical asperity on the order of micrometers onwhich a large electric field is concentrated. Further, the electrospraydevice may provide additional electrode(s) on the ejecting surface towhich electric potential(s) may be applied and controlled independent ofthe electric potentials of the fluid and the substrate in order toadvantageously modify and optimize the electric field for the purpose offocusing the gas phase ions produced by electrospray.

The microchip-based electrospray device of the present inventionprovides minimal extra-column dispersion as a result of a reduction inthe extra-column volume and provides efficient, reproducible, reliableand rugged formation of an electrospray. This electrospray device isperfectly suited as a means of electrospray of fluids frommicrochip-based separation devices. The design of this electrospraydevice is also robust such that the device can be readily mass-producedin a cost-effective, high-yielding process.

In operation, a conductive or partly conductive liquid sample isintroduced into the through-substrate channel entrance orifice on theinjection surface. The liquid is held at a potential voltage, either bymeans of a wire within the fluid delivery channel to the electrospraydevice or by means of an electrode formed on the injection surfaceisolated from the surrounding surface region and from the substrate. Theelectric field strength at the tip of the nozzle is enhanced by theapplication of a voltage to the substrate and/or the ejection surface,preferably zero volts up to approximately less than one-half of thevoltage applied to the fluid. Thus, by the independent control of thefluid/nozzle and substrate/ejection surface voltages, the electrospraydevice of the present invention allows the optimization of the electricfield emanating from the nozzle. The electrospray device of the presentinvention may be placed 1-2 mm or up to 10 mm from the orifice of anatmospheric pressure ionization (API) mass spectrometer to establish astable nanoelectrospray at flow rates as low as 20 nL/min.

The electrospray device may be interfaced or integrated downstream to asampling device, depending on the particular application. For example,the analyte may be electrosprayed onto a surface to coat that surface orinto another device for purposes of conveyance, analysis, and/orsynthesis. As described above with reference to FIGS. 3A-C and 4A-C,highly charged droplets are formed at atmospheric pressure by theelectrospray device from nanoliter-scale volumes of an analyte. Thehighly charged droplets produce gas-phase ions upon sufficientevaporation of solvent molecules which may be sampled, for example,through an ion-sampling orifice of an atmospheric pressure ionizationmass spectrometer (API-MS) for analysis of the electrosprayed fluid.

Multiple Array of Electrospray Devices

One embodiment of the present invention is in the form of a multiplearray of electrospray devices which allows for massive parallelprocessing. The multiple electrospray devices or systems fabricated bymassively parallel processing on a single wafer may then be cut orotherwise separated into multiple devices or systems.

This aspect of the present invention does not have the space constraintsof current piezoelectric dispensers. The nozzles (dispensers) may bepositioned as close as 20 μm allowing for very high-density dispensing.For example, an array of 10,000 dispensing nozzles with a 20 μm outerdiameter and a 50 μm pitch would have an area of 5 mm×5 mm. An array of1,000,000 dispensing nozzles with a 20 μm outer diameter and a 50 μmpitch would have an area of 50 mm×50 mm (or two square inches). Thenumber of dispensing nozzles in an array is only limited by the outerdiameter of the nozzle size chosen and the required spacing for theapplication. FIG. 5A shows a perspective view of a 12-nozzle arrayaligned with an array of receiving wells 152. These receiving wells maybe small volume reservoirs for performing chemical reactions for thepurpose of chemical synthesis, for biological screening or may bethrough-substrate channels for transferring a fluid sample from onemicrochip device to another. FIG. 5B shows a cross-sectional view ofFIG. 5A showing the array in a droplet dispensing mode and the receivingwells 152 depicted as through-substrate channels. Each nozzle 110 has afluid droplet 126 being extracted by an electric field establishedbetween the fluid, substrate 102 and the receiving well plate 154.

The electrospray device may also serve to reproducibly distribute anddeposit a sample from a mother plate to daughter plate(s) bynanoelectrospray deposition or by the droplet method. A chip-basedcombinatorial chemistry system comprising a reaction well block maydefine an array of reservoirs for containing the reaction products froma combinatorially synthesized compound. The reaction well block furtherdefines channels, nozzles and recessed portions such that the fluid ineach reservoir may flow through a corresponding channel and exit througha corresponding nozzle in the form of droplets. The reaction well blockmay define any number of reservoir(s) in any desirable configuration,each reservoir being of a suitable dimension and shape. The volume of areservoir may range from a few picoliters up to several microliters.

The reaction well block may serve as a mother plate to interface to amicrochip-based chemical synthesis apparatus such that the dropletmethod of the electrospray device mays be utilized to reproduciblydistribute discreet quantities of the product solutions to a receivingor daughter plate. The daughter plate defines receiving wells thatcorrespond to each of the reservoirs. The distributed product solutionsin the daughter plate may then be utilized to screen the combinatorialchemical library against biological targets.

The electrospray device may also serve to reproducibly distribute anddeposit an array of samples from a mother plate to daughter plates, forexample, for proteomic screening of new drug candidates. This may be byeither droplet formation or electrospray modes of operation.Electrospray device(s) may be etched into a microdevice capable ofsynthesizing combinatorial chemical libraries. At a desired time, anozzle(s) may apportion a desired amount of a sample(s) or reagent(s)from a mother plate to a daughter plate(s). Control of the nozzledimensions, applied voltages, and time provide a precise andreproducible method of sample apportionment or deposition from an arrayof nozzles, such as for the generation of sample plates for molecularweight determinations by matrix-assisted laser desorption/ionizationtime-of-flight mass spectrometry (“MALDI-TOFMS”). The capability oftransferring analytes from a mother plate to daughter plates may also beutilized to make other daughter plates for other types of assays, suchas proteomic screening. The V_(fluid)/V_(substrate) ratio can be chosenfor formation of an electrospray or droplet mode based on a particularapplication.

An array of electrospray devices can be configured to disperse ink foruse in an ink jet printer. The control and enhancement of the electricfield at the exit of the nozzles on a substrate will allow for avariation of ink apportionment schemes including the formation ofsubmicometer, highly-charged droplets for blending of different colorsof ink.

The electrospray device of the present invention can be integrated withminiaturized liquid sample handling devices for efficient electrosprayof the liquid samples for detection using a mass spectrometer. Theelectrospray device may also be used to distribute and apportion fluidsamples for use with high-throughput screen technology. The electrospraydevice may be chip-to-chip or wafer-to-wafer bonded to plastic, glass,or silicon microchip-based liquid separation devices capable of, forexample, capillary electrophoresis, capillary electrochromatography,affinity chromatography, liquid chromatography (“LC”), or any othercondensed-phase separation technique.

In another aspect of the invention, a microchip-based liquidchromatography device 160 may be provided as shown in FIG. 6A. Theliquid chromatography device generally comprises a separation substrate162 or wafer defining an introduction channel 164 between an entranceorifice and a reservoir 166 and a separation channel 168 between thereservoir and an exit orifice 170. A cover substrate 172 may be bondedto the separation substrate to enclose the reservoir and the separationchannel adjacent to the cover substrate. The separation channel may bepopulated with separation posts 174 as shown in FIG. 6B extending from aside-wall of the separation channel perpendicular to the fluid flowthrough the separation channel. Preferably, the separation posts arecoplanar or level with the surface of the separation substrate such thatthey are protected against accidental breakage during the manufacturingprocess. Component separation occurs in the separation channel where theseparation posts perform the liquid chromatography function by providinga large surface area for the interaction of fluid flowing through theseparation channel.

The liquid chromatography device may be integrated with an electrospraydevice such that the exit orifice of the liquid chromatography deviceforms a homogenous interface with the entrance orifice of theelectrospray device. This allows the on-chip delivery of fluid from theliquid chromatography device to the electrospray device to generate anelectrospray. The nozzle, channel, and recessed portion of theelectrospray device may be etched from the substrate of the liquidchromatography device. FIG. 6C is a cross-sectional view of FIG. 6Bwherein the exit orifice 170 of the liquid chromatography device is thethrough-substrate channel 104 of an electrospray device. The liquidchromatography device may further comprise one or more electrodes 176for application of electric potentials to the fluid at locations alongthe fluid path. The application of different electric potentials alongthe fluid path may facilitate the fluid flow through the fluid pathusing the electrophoretic properties of the fluid and chemical speciescontained therein. Also shown are the electrospray nozzle 110, recessedannular region 114, and the electrospray controlling electrodes 122 onthe ejection surface 112 of the substrate.

The introduction 164 and separation 168 channels, the entrance and exit170 orifices, and the separation posts 174 are preferably etched from asilicon substrate by reactive-ion etching and other standardsemiconductor processing techniques. The separation posts are preferablyoxidized silicon posts 174′ to electrically insulate the posts andchannel from the silicon substrate. A silicon dioxide layer 118 may begrown on all surfaces of the separation substrate 162. Silicon nitride120 may be further deposited on the silicon dioxide to provide amoisture barrier and prevent diffusion of water and ions to thesubstrate. The surface of the silicon posts may be further chemicallymodified to form a stationary phase to optimize the interaction of thecomponents of the sample fluid with the stationary separation posts.

Photolithography and reactive-ion etching limit the layout design ofseparation post diameters and inter-post spacing to approximately 1 μm.However, because the thermal oxidation process consumes approximately0.46 μm of silicon to form each micrometer of silicon dioxide, thethermal oxidation process results in a volumetric expansion. Thisvolumetric expansion may be utilized to reduce the spacing between theseparation posts to sub-micrometer dimensions as shown in FIG. 7. Forexample, if the final layout is a channel populated with 1 μm silicondioxide posts separated by 0.5 μm, the following method may be used togenerate such a device. If the layout begins with 1 μm silicon posts 180spaced by 0.5 μm (FIG. 7A.), oxidizing the silicon posts using anelevated temperature, oxidizing furnace until the post diameters reached1.5 μm would consume 0.12 μm of silicon (FIG. 7B.). The silicon dioxide182 that was formed can be removed by placing the silicon substrate in ahydrofluoric acid solution. The hydrofluoric acid will selectivelyremove the silicon dioxide from the silicon substrate. The remainingsilicon posts would now have a diameter of 0.77 μm (FIG. 7C.). If thesilicon posts were oxidized to 1.44 μm diameter, 0.31 μm of siliconwould be consumed (FIG. 7D.). Removal of the silicon dioxide would leavesilicon posts of 0.46 μm diameter (FIG. 7E.). Complete oxidation of the0.46 μm silicon posts 180 will result in the formation of 1 μm silicondioxide posts 182 spaced by 0.5 μm (FIG. 7F.). Further, because theoxidation process is well-controlled, separation post dimensions,including the inter-post spacing, in the sub-micrometer regime can beformed reproducibly and in a high yielding manner.

FIGS. 8A and 8B show plan views of a computer-aided design (CAD) layout190 of a 50 μm wide channel 192 containing 1 μm silicon posts spaced by0.5 μm. The black squares 194 represent unexposed areas of the channelwhile the gray 196 areas represent exposed areas of the channel. Theexposed areas are removed during the silicon processing of thesubstrate, while the unexposed areas remain. The result of theprocessing is a channel etched to a depth of a few tens of micrometerscontaining 1 μm silicon posts spaced by 0.5 μm. The remaining siliconsubstrate can then be further oxidized in an oxidizing furnace to growthe silicon dioxide layer to any required thickness without affectingthe completely oxidized silicon posts. Further processing of the siliconsubstrate such as LPCVD of silicon nitride and/or LPCVD or plasmaenhanced chemical vapor deposition (“PECVD”) of silicon dioxide ispossible.

An array or matrix of multiple electrospray devices of the presentinvention may be manufactured on a single microchip as siliconfabrication using standard, well-controlled thin-film processes. Thisnot only eliminates handling of such micro components but also allowsfor rapid parallel processing of functionally alike elements. Thenozzles may be radially positioned, for example, about a circle having arelatively small diameter near the center of the chip. Thus, a 96 radialarray of electrospray devices of the present invention may be positionedin front of an electrospray mass spectrometer with no requirement tomove or reposition the microchip. This radial design providessignificant advantages of time and cost efficiency, control, andreproducibility when analyzing multiple channels by electrospray massspectrometry. The low cost of these electrospray devices allows forone-time use such that cross-contamination from different liquid samplesmay be eliminated.

The requirement to minimize the variations in the cross-sectional areaalong the length of a separation channel also applies when combining aseparation device with an electrospray device. The cross-sectional areafor the inner diameter of the nozzle, Nozzle_(Area) ², of anelectrospray device should be approximately the same as the channelcross-sectional area, Channel_(Area) ². In practice, a Nozzle_(Area)²/Channel_(Area) ² ratio less than 2 is desirable. The cross-sectionalarea of a separation device can be determined by calculating thepercentage of cross-sectional area for a post and the separation from anadjacent post. The cross-sectional area for a given channel can then becalculated from the following equation:

Channel_(Area) _(²)=Width_(Ch)*Depth_(Ch)(1−(Dia_(post)/(Diam_(post)+Spacing_(post)))  (4)

where Width_(Ch) is the separation channel width, Depth_(Ch) is theseparation channel depth, Dia_(post) is the post diameter andSpacing_(post) is the post spacing. Setting the cross-sectional area ofthe electrospray nozzle equal to the cross-sectional area of theseparation channel allows the calculation for the optimum inner diameterfor the electrospray device for a particular separation channel layout.The cross-sectional area for a cylindrical nozzle, Nozzle_(Area) ² isdefined by equation 5:

Nozzle_(Area) _(²) =πr ²=π(d/2)²  (5)

where r is the inner radius and d is the inner diameter of the nozzle.

Setting the Nozzle_(Area) ² equal to the Channel_(Area) ² allows thedetermination of the optimum nozzle inner diameter, Nozzle_(Inner Dia),for a given channel cross-sectional area from equation 6:$\begin{matrix}{{Nozzle}_{InnerDia} = {2*\sqrt{\frac{{Channel}_{{Area}^{2}}}{\pi}}}} & (6)\end{matrix}$

Table 1 lists some examples of the optimum nozzle inner diameter forsome examples of posts diameters and spacings for a 50 μm wide by 10 μmdeep channel.

TABLE 1 Relationship between a 50 μm width by 10 μm depth channelpopulated with posts and the optimum electrospray nozzle inner diameterPost Post Channel and Nozzle Electrospray Nozzle Diameter, Spacing,Cross-sectional Areas, Inner Diameter μm μm μm² μm 1 0.1 45 7.6 1 0.2 8310.3 1 0.3 115 12.1 1 0.4 143 13.5 1 0.5 167 14.6 1 0.8 222 16.8 1 1 25017.8 1 1.5 300 19.5 2 0.1 24 5.5 2 0.2 45 7.6 2 0.3 65 9.1 2 0.4 83 10.32 0.5 100 11.3 2 0.8 143 13.5 2 1 167 14.6 2 1.5 214 16.5

In yet another aspect of the present invention, multiples of the liquidchromatography-electrospray system may be formed on a single chip todeliver a multiplicity of samples to a common point for subsequentsequential analysis. The multiple nozzles of the electrospray devicesmay be radially positioned about a circle having a relatively smalldiameter near the center of the single chip.

A radially distributed array of electrospray nozzles on a multi-systemchip may be interfaced with an ion-sampling orifice of an electrospraymass spectrometer by positioning the nozzles near the ion-samplingorifice. A tight radial configuration of the electrospray nozzles allowsthe positioning thereof in close proximity to the ion-sampling orificeof an electrospray mass spectrometer. For example, 96 20 μm nozzles maybe etched around a 1 mm radius circle with a separation of 65 μm.

A multi-system chip thus provides a rapid sequential chemical analysissystem fabricated using microelectromechanical systems (“MEMS”)technology. For example, the multi-system chip enables automated,sequential separation and injection of a multiplicity of samples,resulting in significantly greater analysis throughput and utilizationof the mass spectrometer instrument for, for example, high-throughputdetection of compounds for drug discovery.

Another aspect of the present invention provides a siliconmicrochip-based electrospray device for producing electrospray of aliquid sample. The electrospray device may be interfaced downstream toan atmospheric pressure ionization mass spectrometer (“API-MS”) foranalysis of the electrosprayed fluid. Another aspect of the invention isan integrated miniaturized liquid phase separation device, which mayhave, for example, glass, plastic or silicon substrates integral withthe electrospray device.

Electrospray Device Fabrication Procedure

The electrospray device 100 is preferably fabricated as a monolithicsilicon substrate utilizing well-established, controlled thin-filmsilicon processing techniques such as thermal oxidation,photolithography, reactive-ion etching (RIE), chemical vapor deposition,ion implantation, and metal deposition. Fabrication using such siliconprocessing techniques facilitates massively parallel processing ofsimilar devices, is time- and cost-efficient, allows for tighter controlof critical dimensions, is easily reproducible, and results in a whollyintegral device, thereby eliminating any assembly requirements. Further,the fabrication sequence may be easily extended to create physicalaspects or features on the injection surface and/or ejection surface ofthe electrospray device to facilitate interfacing and connection to afluid delivery system or to facilitate integration with a fluid deliverysub-system to create a single integrated system.

Injection Surface Processing: Entrance to Through-Wafer Channel

FIGS. 9A-9E illustrate the processing steps for the injection side ofthe substrate in fabricating the electrospray device of the presentinvention. Referring to the plan and cross-sectional views,respectively, of FIGS. 9A and 9B (a cross-sectional view taken alongline 9B—9B of FIG. 9A), a double-side polished silicon wafer 200 issubjected to an elevated temperature in an oxidizing environment to growa layer or film of silicon dioxide 204 on the injection side 203 and alayer or film of silicon dioxide 206 on the ejection side 205 of thesubstrate 202. Each of the resulting silicon dioxide layers 204, 206 hasa thickness of approximately 1-2 μm. The silicon dioxide layers 204, 206serve as masks for subsequent selective etching of certain areas of thesilicon substrate 202. The silicon dioxide layer 206 also serves as anetch stop for the through-substrate channel etch as described below.

A film of positive-working photoresist 208 is deposited on the silicondioxide layer 204 on the injection side 203 of the substrate 200. Anarea of the photoresist 208 corresponding to the entrance to athrough-wafer channel which will be subsequently etched is selectivelyexposed through a mask by an optical lithographic exposure tool passingshort-wavelength light, such as blue or near-ultraviolet at wavelengthsof 365, 405, or 436 nanometers.

As shown in the cross-sectional views of FIGS. 9C and 9D, afterdevelopment of the photoresist 208, the exposed area 210 of thephotoresist is removed and open to the underlying silicon dioxide layer204, while the unexposed areas remain protected by photoresist 208′. Theexposed area 212 of the silicon dioxide layer 204 is then etched by afluorine-based plasma with a high degree of anisotropy and selectivityto the protective photoresist 208′ until the silicon substrate 202 isreached. As shown in the cross-sectional view of FIG. 9D, the remainingphotoresist 208′ provides additional masking during a subsequentfluorine based silicon etch to vertically etch certain patterns into theinjection side 203 of the silicon substrate 200.

As shown in the cross-sectional view of FIG. 9E, the through-substratechannel 214 in the silicon substrate is vertically etched by anotherfluorine-based etch. An advantage of the fabrication process describedherein is that the dimensions of the through channel, such as the aspectratio (i.e. depth to width), can be reliably and reproducibly limitedand controlled. The through-substrate channel is selectively etchedthrough the silicon substrate until the silicon dioxide layer on theejection surface is reached.

The through-substrate channel is used to align the ejection surfacestructures with the injection surface through-wafer channels. Thethrough-substrate channel is etched through the substrate to the silicondioxide layer 206 on the ejection side 205 of the substrate 200. Thissilicon dioxide layer 206 on the ejection surface serves as an etch stopfor the injection surface processing. Silicon dioxide is transparent tovisible light which allows the alignment of the injection side etch withthe ejection side mask. This alignment scheme allows for alignment ofinjection and ejection side features to within 1 μm. The silicon dioxidelayer on the ejection surface is still intact and provides for easycoating of resist on the ejection side for the subsequent ejectionsurface processing.

Ejection Surface Processing: Nozzle and Surrounding Surface Structure

FIGS. 10A-10F illustrate the processing steps for the ejection side 205of the substrate 202 in fabricating the electrospray device 100 of thepresent invention. As shown in the cross-sectional view in FIG. 10B (across-sectional view taken along line 10B—10B of FIG. 10A), a film ofpositive-working photoresist 216 is deposited on the silicon dioxidelayer 206 on the ejection side 205 of the substrate 202. Patterns on theejection side 205 are aligned to those previously formed on theinjection side 203 of the substrate 202 using a through-substratealignment mark.

After alignment, areas of the photoresist 216 that define the outerdiameter of the nozzle and the outer diameter of the recessed annularregion are selectively exposed through an ejection side mask by anoptical lithographic exposure tool passing short-wavelength light, suchas blue or near-ultraviolet at wavelengths of 365, 405, or 436nanometers. As shown in the cross-sectional view of FIG. 10C, thephotoresist 216 is then developed to remove the exposed areas of thephotoresist 218 such that the recessed annular region is open to theunderlying silicon dioxide layer 220, while the unexposed areas remainprotected by photoresist 216′. The exposed area 220 of the silicondioxide layer 206 is then etched by a fluorine-based plasma with a highdegree of anisotropy and selectivity to the protective photoresist 216′until the silicon substrate 202 is reached as shown in FIG. 10D.

As shown in FIG. 10E, a fluorine-based etch creates a recessed annularregion 222 that defines an ejection nozzle 224. After the desired depthis achieved for defining the recessed annular region 222 and nozzle 224,the remaining photoresist 216′ is then removed in an oxygen plasma or inan actively oxidizing chemical bath like sulfuric acid (H₂SO₄) activatedwith hydrogen peroxide (H₂O₂). Then, the silicon dioxide layer 206 isremoved using hydrofluoric acid to open up the through-substrate channel214 as shown in FIG. 10F.

The fabrication method confers superior mechanical stability to thefabricated electrospray device by etching the features of theelectrospray device from a monocrystalline silicon substrate without anyneed for assembly. The alignment scheme allows for nozzle walls of lessthan 2 μm and nozzle outer diameters down to 5 μm to be fabricatedreproducibly. The fabrication sequence allows for the control of thenozzle height by adjusting the relative amounts of ejection side siliconetching. Further, the lateral extent and shape of the recessed annularregion can be controlled independently of its depth. The depth of therecessed annular region also determines the nozzle height and isdetermined by the extent of the etch on the ejection side of thesubstrate. Control of the lateral extent and shape of the recessedannular region provides the ability to modify and control the electricfield between the electrospray device and an extracting electrode.

Alternatively, the fabrication of the electrospray device may beaccomplished whereby the through-substrate channel is etched partly fromeach side of the substrate in two steps in combination with athrough-substrate alignment mark as shown in FIGS. 11A to B and 12A-E.

Injection Surface Processing: Entrance to Through-Wafer Channel

FIGS. 11A-11D illustrate the processing steps for the injection side ofthe substrate in fabricating the electrospray device of the presentinvention. Referring to the plan and cross-sectional views,respectively, of FIGS. 11A and 11B (taken along line 11B—11B of FIG.11B), a double-side polished silicon substrate 200 is subjected to anelevated temperature in an oxidizing environment to grow a layer or filmof silicon dioxide 204 on the injection side 203 and a layer or film ofsilicon dioxide 206 on the ejection side 205 of the substrate 200. Eachof the resulting silicon dioxide layers 204 and 206 has a thickness ofapproximately 1-2 μm. The silicon dioxide layers 204 and 206 serve asmasks for subsequent selective etching of certain areas of the siliconsubstrate. The silicon dioxide layer 206 also serves as an etch stop forthe through-substrate alignment feature as described below.

A film of positive-working photoresist 208′ is deposited on the silicondioxide layer 204 on the injection side 203 of the substrate 200. Anarea of the photoresist corresponding to the through wafer alignment 208and the device channels 202 which will be subsequently etched isselectively exposed through a mask by an optical lithographic exposuretool passing short-wavelength light, such as blue or near-ultraviolet atwavelengths of 365, 405, or 436 nanometers. After development of thephotoresist 208′, the exposed area of the photoresist is removed and theunderlying silicon dioxide layer of the alignment 210 and device 212channels is exposed. The unexposed areas remain protected by theunexposed photoresist 208′. As shown in FIG. 11C the exposed area 212 ofthe silicon dioxide layer 204 is then etched by a fluorine-based plasmawith a high degree of anisotropy and selectivity to the protectivephotoresist 208′ until the silicon substrate 200 is reached. Theremaining photoresist 208′ provides additional masking during asubsequent fluorine based silicon etch to vertically etch certainpatterns into the injection side 203 of the silicon substrate 204.

As shown in the cross-sectional view of FIG. 11D, the through-substratealignment channel 215 and injection side channel 211 in the siliconsubstrate 200 is vertically etched by another fluorine-based etch. Anadvantage of the fabrication process described herein is that thedimensions of the features to be etched in silicon, such as the aspectratio (depth to width), can be reliably and reproducibly limited andcontrolled. The fluorine-based etch rate is dependent on the featuredimensions being etched. Therefore, larger features etch more quicklythrough a substrate than smaller features. For the process describedhere, the through-substrate alignment mark 215 may be slightly larger insize (diameter) than the injection side channel 211. Therefore, thelarger diameter through-substrate alignment channel 215 etches morequickly through the substrate 202 than the injection side channel 211.The through-substrate alignment marks are selectively etched completelythrough the silicon substrate 202 until the silicon dioxide layer 206,serving as an etch stop on the ejection surface 205, is reached.However, the smaller diameter injection side channel 211 is only etchedpartially through the wafer. Typically, the through-substrate alignmentmark may be equivalent in diameter to up to tens of microns larger thanthe final through-substrate channel 214 to provide the requiredalignment tolerances.

The through-substrate alignment mark, consisting of for example, a 25 μmdiameter circle, is incorporated in the channel mask. Thethrough-substrate alignment mark is etched through the substrate to thesilicon dioxide layer on the ejection side of the substrate. Thissilicon dioxide layer on the ejection surface serves as an etch stop forthe injection surface processing. Silicon dioxide is transparent tovisible light which allows the alignment mark from the injection sideetch to be aligned with the ejection side mask. This alignment schemeallows for alignment of injection and ejection side features to within 1μm. The silicon dioxide layer on the ejection surface is still intactand provides for easy coating of resist on the ejection side for thesubsequent ejection surface processing.

Ejection Surface Processing: Nozzle and Surrounding Surface Structure

FIGS. 12A-E illustrate the processing steps for the ejection side 205 ofthe substrate in fabricating the electrospray device 100 of the presentinvention. Referring to the plan and cross-sectional views,respectively, of FIGS. 12A and 12B (taken along line 12B—12B of FIG.12A), a film of positive-working photoresist is deposited on the silicondioxide layer 206 on the ejection side 205 of the substrate 202.Patterns that define the inner and outer diameter of the nozzle and theouter diameter of the recessed annular region on the ejection side 205are aligned to those previously formed on the injection side 203 of thesubstrate using the through-substrate alignment channels 215.

After alignment, areas of the photoresist that define the inner andouter diameter of the nozzle and the outer diameter of the recessedannular region are selectively exposed through an ejection side mask byan optical lithographic exposure tool. As shown in the cross-sectionalview of FIG. 12C, the exposed photoresist 218′ is then developed toremove the exposed areas of the photoresist such that the devicefeatures 212 are open to the underlying silicon dioxide layer 206, whilethe unexposed areas remain protected by the unexposed photoresist 218′.The exposed areas 212 of the silicon dioxide layer 206 are then etchedby a fluorine-based plasma with a high degree of anisotropy andselectivity to the protective photoresist 218′ until the siliconsubstrate 200 is reached as seen in FIG. 12C.

As shown in FIG. 12D, a fluorine-based etch creates an ejection nozzle224, a recessed annular region 222 exterior to the nozzle and anejection side channel 213 that is etched until the injection sidechannel 211 is reached forming the through-substrate channel 214. Afterthe desired depth for the recessed annular region 222 and the nozzle 224are achieved, the remaining photoresist 218′ is then removed in anoxygen plasma or in an actively oxidizing chemical bath like sulfuricacid (H₂SO₄) activated with hydrogen peroxide (H₂O₂). The silicondioxide layers 204 and 206 are removed using hydrofluoric acid to openup the through-substrate channel 214 as shown in FIG. 12E.

This fabrication sequence confers superior mechanical stability to thefabricated electrospray device by etching the features of theelectrospray device from a monocrystalline silicon substrate without anyneed for assembly. Further, use of a visible alignment mark as describedin the fabrication sequence of this device allows for alignment ofinjection side and ejection side features to better than 1 μm. Thisallows for overall nozzle dimensions that are smaller than previouslyachieved that use prior disclosed alignment schemes using infraredlight. Control of the lateral extent and shape of the recessed annularregion provides the ability to modify and control the electric fieldbetween the electrospray device 100 and an extracting electrode.

Discussed below is another scheme for fabricating a through waferchannel and nozzle. Here, front side to backside alignment of thechannel and nozzle is conducted by patterning both injection andejection sides of the wafer together prior to the etch processing. Adouble-side polished silicon substrate is subjected to an elevatedtemperature in an oxidizing environment to grow a layer or film ofsilicon dioxide on the injection and ejection side of the substrate. Theresulting silicon dioxide layer has a thickness of approximately 1-2 μm.The silicon dioxide layer serves as a mask for subsequent selectiveetching of certain areas of the silicon substrate. A film ofpositive-working photoresist is deposited on the silicon dioxide layerof the injection and ejection sides of the wafer.

The injection and ejection masks are aligned to each other using anoptical lithographic exposure tool. The silicon substrate is positionedbetween the aligned masks followed by injection and ejection sideexposure by an optical lithographic exposure tool. Subsequent processingof the wafer is conducted as described previously.

Preparation of the Substrate for Electrical Isolation

As shown in the cross-sectional views of FIGS. 13-15, a layer of silicondioxide 117 is grown on all silicon surfaces of the substrate 102 bysubjecting the silicon substrate to elevated temperature in an oxidizingambient. This layer is grown to typically less than 1 μm to remove anymaterials from the surfaces of the substrate. This silicon dioxide layeris removed from the silicon substrate using hydrofluoric acid. Thesilicon substrate is further subjected to elevated temperature in anoxidizing ambient furnace to grow silicon dioxide 118 to a thickness of1 to 4 μm. A layer of silicon nitride 120 is further deposited on top ofthe silicon dioxide layer using low pressure chemical vapor deposition(“LPCVD”) providing a conformal coating of silicon nitride on allsurfaces up to 2 μm in thickness. The silicon nitride prevents water andions from penetrating through the silicon dioxide layer, causing anelectrical connection between the fluid in the through-wafer channel 104and the silicon substrate 102. The layers of silicon dioxide 118 andsilicon nitride 120 over all surfaces of the substrate, electricallyisolates a fluid in the channel 104 from the silicon substrate andpermits the application and sustenance of different electricalpotentials to the fluid in the channel 104 and to the silicon substrate102.

All silicon surfaces are oxidized to form silicon dioxide with athickness that is controllable through choice of temperature and time ofoxidation. All silicon dioxide surfaces are LPCVD coated with siliconnitride. The final thickness of the silicon dioxide and silicon nitridecan be selected to provide the desired degree of electrical isolation inthe device. A thicker layer of silicon dioxide and silicon nitrideprovides a greater resistance to electrical breakdown. The siliconsubstrate 100 is divided into the desired size or array of electrospraydevices for purposes of metalization of the edge of the siliconsubstrate. As shown in FIG. 15, the edge 124 of the silicon substrate iscoated with a conductive material 122 using well known thermalevaporation and metal deposition techniques.

The above described fabrication sequence for the electrospray device 100can be easily adapted to and is applicable for the simultaneousfabrication of a single monolithic system comprising multipleelectrospray devices including multiple channels and/or multipleejection nozzles embodied in a single monolithic substrate. Further, theprocessing steps may be modified to fabricate similar or differentelectrospray devices merely by, for example, modifying the layout designand/or by changing the polarity of the photomask and utilizingnegative-working photoresist rather than utilizing positive-workingphotoresist.

Liquid Chromatography and Electrospray Device Fabrication Procedure

The fabrication of a liquid chromatography/electrospray (“LC/ESI”)device of the present invention is explained with reference to FIGS.16A-I. The LC/ESI device is preferably fabricated as a monolithicsilicon micro device utilizing established, well-controlled thin-filmsilicon processing techniques such as thermal oxidation,photolithography, reactive-ion etching (RIE), chemical vapor deposition,ion implantation, and metal deposition. Fabrication using such siliconprocessing techniques facilitates massively parallel processing ofsimilar devices, is time- and cost-efficient, allows for tighter controlof critical dimensions, is easily reproducible. and results in a whollyintegral device, thereby eliminating any assembly requirements.

Referring to the plan and cross-sectional views, respectively, of FIGS.16A and 16B (taken along line 16B—16B of FIG. 16A), a silicon wafersubstrate 500, double-side polished and approximately 250-300 μm inthickness, is subjected to an elevated temperature in an oxidizingambient to grow a layer or film of silicon dioxide 502 on thechromatography side 503 and a layer or film of silicon dioxide 504 onthe electrospray side 505 of the separation substrate 500. Each of theresulting silicon dioxide layers 502 and 504 has a thickness ofapproximately 1-2 μm. The silicon dioxide layers 502 and 504 serve asmasks for subsequent selective etching of certain areas of theseparation substrate 500.

A film of positive-working photoresist 506 is deposited on the silicondioxide layer 502 on the chromatography side 503 of the separationsubstrate 500. Certain areas of the photoresist 506 corresponding to thereservoirs, sample injection channels, separation channel and separationposts which will be subsequently etched are selectively exposed througha mask by an optical lithographic exposure tool.

Referring to the cross-sectional view of FIG. 16C, after development ofthe photoresist 506, the exposed areas of the photoresist correspondingto the reservoir 508 and separation channel 510, respectively, areremoved and open to the underlying silicon dioxide layer 502, while theunexposed areas remain protected by photoresist 506′. The exposed areas508 and 510 of the silicon dioxide layer 502 are then etched by afluorine-based plasma with a high degree of anisotropy and selectivityto the protective photoresist 506′ until the silicon separationsubstrate 500 is reached. The remaining photoresist is removed in anoxygen plasma or in an actively oxidizing chemical bath like sulfuricacid (H₂SO₄) activated with hydrogen peroxide (H₂O₂).

As shown in the cross-sectional view of FIG. 16D, the reservoir 410, theseparation channel 412, and the separation posts 416 in the separationchannel are vertically formed in the silicon separation substrate 500 byanother fluorine-based etch as described in U.S. Pat. No. 5,501,893,which is hereby incorporated by reference. Preferably, the reservoir 410and the separation channel 412 have the same depth controlled by theetch time at a known etch rate. The depth of the reservoir 410 and thechannel 412 is preferably between approximately 5-20 μm and morepreferably approximately 10-15 μm.

Referring to the cross-sectional view of FIG. 16E, the remainingphotoresist 506′ is removed and the substrate 500 is subjected to anelevated temperature in an oxidizing ambient to grow a layer or film ofsilicon dioxide 502′ sufficient to minimize the space between the posts416 created during the previous etch described in FIG. 16D.Alternatively, PECVD silicon dioxide may be deposited on thechromatography side of the substrate sufficient to enclose the spacebetween the posts 416.

Referring to the cross-sectional view of FIG. 16F, a film ofpositive-working photoresist 516 is deposited on the silicon dioxidelayer 502′ on the chromatography side 503 of the separation substrate500. Referring now to the plan and cross-sectional views of FIGS. 16Gand 16H (taken along line 16H—16H of the FIG. 16G), respectively,certain areas of the photoresist 516 corresponding to the reservoirthrough-substrate channel 404 and the electrospray through-substratechannel 406 that will be subsequently etched are selectively exposedthrough a mask by an optical lithographic exposure tool. Afterdevelopment of the photoresist 516′, the exposed area 518 of thephotoresist 516′ corresponding to the reservoir through-substratechannel and the electrospray through-substrate channel is removed toexpose the underlying silicon dioxide layer 502′ of the separationsubstrate 500. The exposed silicon dioxide layer is then etched by afluorine-based plasma with a high degree of anisotropy and selectivityto the protective photoresist 516′ until the silicon separationsubstrate 500 is reached. The remaining photoresist is left in place toprovide additional masking during the subsequent through-substrate etchof the silicon substrate 500.

Referring now to the cross-sectional view of FIG. 16I, the reservoirthrough-substrate channel and the electrospray through-substrate channelis vertically formed through the silicon separation substrate 500 by afluorine-based etch as described in U.S. Pat. No. 5,501,893, which ishereby incorporated by reference. The reservoir through-substratechannel 404 and the electrospray through-substrate channel 406 areetched until the silicon dioxide layer 504 is reached. The remainingphotoresist is removed in an oxygen plasma or in an actively oxidizingchemical bath like sulfuric acid (H₂SO₄) activated with hydrogenperoxide (H₂O₂).

The remaining nozzle and recessed annular region are etched using thesame method as that outlined previously in the fabrication of theejection surface processing of the electrospray device as shown in FIGS.17A-D. FIG. 17A is a plan view of the pattern that defines the recessedannular region 408 on the electrospray side 505 of the substrate 500.The existing features are aligned to those previously formed on thechromatography side 503 of the substrate using through-substratealignment channels.

After alignment, areas of the photoresist that define the pattern thatdefines the recessed annular region 408 on the electrospray side 505 ofthe substrate 500 are selectively exposed through an ejection side maskby an optical lithographic exposure tool. As shown in thecross-sectional view of FIG. 17B (taken along line 17B—17B of FIG. 17A),the exposed photoresist 518′ is then developed to remove the exposedareas of the photoresist to the underlying silicon dioxide layer 504.The exposed areas of the silicon dioxide layer 504 are then etched by afluorine-based plasma with a high degree of anisotropy and selectivityto the protective photoresist 518′ until the silicon substrate 500 isreached.

As shown in FIG. 17C, a fluorine-based etch creates an ejection nozzle424, a recessed annular region 422 exterior to the nozzle. After thedesired depth for the recessed annular region 422 and the nozzle 424 areachieved, the remaining photoresist 518′ is then removed in an oxygenplasma or in an actively oxidizing chemical bath like sulfuric acid(H₂SO₄) activated with hydrogen peroxide (H₂O). The silicon dioxidelayers 502′ and 504 are removed using hydrofluoric acid to open up thethrough-substrate channel as shown in FIG. 17D.

An advantage to defining the reservoir through-substrate channel and theelectrospray nozzle on the same side of the completed LC/ESI device isthat the backside of the substrate is then free from any features. Thissubstrate may be bonded to another glass or silicon substrate that maybe further bonded to a protective package.

Preparation of the Substrate for Electrical Isolation

A layer of silicon dioxide is grown on all silicon surfaces of thesubstrate by subjecting the silicon substrate to elevated temperature inan oxidizing ambient. This layer is grown to typically less than 1 μm toremove any materials from the surfaces of the substrate. This silicondioxide layer is removed from the silicon substrate using hydrofluoricacid. The silicon substrate is further subjected to elevated temperaturein an oxidizing ambient to grow silicon dioxide 118 to a thickness of 1to 4 μm. A layer of silicon nitride 122 is further deposited on top ofthe silicon dioxide layer using low pressure chemical vapor deposition(LPCVD) providing a conformal coating of silicon nitride on all surfacesup to 2 μm in thickness. Alternatively, plasma enhanced chemical vapordeposition can be used to selectively deposit silicon dioxide and/orsilicon nitride on vertical surfaces exposed to the plasma. Siliconnitride is well known to prevent water and ions from penetrating througha silicon dioxide layer of silicon devices. The silicon nitride furtherprevents an electrical connection between the fluid in the LC/ESI deviceand the silicon substrate 162. The layer of silicon dioxide 118 andsilicon nitride 122 over all surfaces of the silicon substrate 162electrically isolates a fluid in the channel from the substrate 162 andpermits the application and sustenance of different electricalpotentials to the fluid in the device and to the silicon substrate 200.Additional layers of silicon dioxide can be deposited using LPCVD toallow for chemical modification of silanol groups on the silicon dioxidesurface. The final cross-sectional area should be identical along theentire length of the separation channel and the electrospraythrough-substrate channel.

Electrodes 176 and bond pads in the cover substrate, preferablycomprising glass and/or silicon, are deposited using similar well-knownthermal evaporation and metal deposition. The cover substrate ispreferably hermetically bonded by any suitable method to the separationsubstrate for containment and isolation of the fluid in the LC/ESIdevice. Critical considerations in any bonding method include thealignment of features in the separation and the cover substrates toensure proper functioning of the liquid chromatography device afterbonding and the provision in layout design for conductive lead-throughssuch as the bond pads and/or metal lines so that the electrodes (if any)are accessible from outside the liquid chromatography device.

The cross-sectional schematic view of FIG. 6C shows a liquidchromatography-electrospray system 160 comprising a liquidchromatography device of the present invention integrated with anelectrospray device of the present invention. A homogeneous interface isformed between the exit orifice 170 of the liquid chromatography deviceand the entrance orifice of the electrospray device. The singleintegrated system allows for the fluid exiting the exit orifice of theliquid chromatography device to be delivered on-chip to the entranceorifice of the electrospray device in order to generate an electrospray.

Multiple Liquid Chromatography-Electrospray Systems on a Single Chip

Multiples of the liquid chromatography-electrospray system may be formedon a single chip to deliver a multiplicity of samples to a common pointfor subsequent sequential analysis.

Interface of a Multi-System Chip to Mass Spectrometer

A radially distributed array of electrospray nozzles on a multi-systemchip may be interfaced with a sampling orifice of a mass spectrometer bypositioning the nozzles near the sampling orifice. The tight radialconfiguration of electrospray nozzles allows the positioning thereof inclose proximity to the sampling orifice of a mass spectrometer.

A multi-system chip may be rotated relative to the sampling orifice toposition one or more of the nozzles for electrospray near the samplingorifice. Appropriate voltage(s) may then be applied to the one or moreof the nozzles for electrospray. Alternatively, the multi-system chipmay be fixed relative to the sampling orifice of a mass spectrometersuch that all nozzles, which converge in a relatively tight radius, areappropriately positioned for the electrospray process. As is evident,eliminating the need for nozzle repositioning allows for highlyreproducible and quick alignment of the single multi-system chip andincreases the speed of the analyses.

Although the invention has been described in detail for the purpose ofillustration, it is understood that such detail is solely for thatpurpose, and variations can be made therein by those skilled in the artwithout departing from the spirit and scope of the invention which isdefined by the following claims.

What is claimed:
 1. An electrospray device comprising: a substratehaving an injection surface and an ejection surface opposing theinjection surface, wherein the substrate is an integral monolithcomprising: an entrance orifice on the injection surface; an exitorifice on the ejection surface; a channel extending between theentrance orifice and the exit orifice; a recess extending into theejection surface and surrounding the exit orifice, thereby defining anozzle on the ejection surface; and an electric field application systemdefining an electric field consisting essentially of: a first electrodeimparting a first electrical potential to fluid passing through theelectrospray device and a second electrode substantially co-planar withthe exit orifice imparting a second electrical potential, wherein thefirst and the second electrodes are positioned to define an electricfield surrounding the exit orifice.
 2. The electrospray device accordingto claim 1, wherein the second electrode is located on the ejectionsurface of the substrate.
 3. The electrospray device according to claim2, wherein the substrate is non-conductive.
 4. The electrospray deviceaccording to claim 3, wherein the substrate is glass.
 5. Theelectrospray device according to claim 3, wherein the substrate ispolymeric.
 6. The electrospray device according to claim 2, wherein thesubstrate is conductive and insulated from the fluid.
 7. Theelectrospray device according to claim 6, wherein the substrate issilicon.
 8. The electrospray device according to claim 6, wherein thesubstrate is polymeric.
 9. The electrospray device according to claim 6,wherein the injection surface, the ejection surface, and the channelextending between the entrance orifice on the injection surface and theexit orifice on the ejection surface are coated with an insulatinglayer.
 10. The electrospray device according to claim 1, wherein thesecond electrode is the substrat and is insulated from fluid.
 11. Theelectrospray device according to claim 1, wherein the second electrodeis not integral with the ejection surface.
 12. The electrospray deviceaccording to claim 1, wherein application of potentials to the first andsecond electrodes causes fluid passing through the electrospray deviceto discharge from the exit orifice in the form of a spray.
 13. Theelectrospray device according to claim 1, wherein application ofpotentials to the first and second electrodes causes fluid passingthrough the electrospray device to discharge from the exit orifice inthe form of droplets.
 14. The electrospray device according to claim 1,wherein the substrate has a plurality of entrance orifices on theinjection surface, a plurality of exit orifices on the ejection surfacewith each of the plurality of exit orifices corresponding to arespective one of the plurality entrance orifices, and a plurality ofchannels extending between one of the plurality of exit orifices and thecorresponding one of the plurality of entrance orifices.
 15. Theelectrospray device according to claim 14, further comprising aplurality of first and second electrode pairs corresponding to arespective one of each of the plurality of exit orifices.
 16. Theelectrospray device according to claim 14, further comprising a conduitpositioned to provide fluid to an entrance orifice.
 17. The electrospraydevice according to claim 16, wherein the conduit is conductive.
 18. Theelectrospray device according to claim 17, wherein the conduit is acapillary, micropipette, or microchip.
 19. The electrospray deviceaccording to claim 16, wherein the conduit is configured to achieve aseal with the entrance orifice.
 20. The electrospray device according toclaim 19, wherein the conduit is repositioned to seal another entranceorifice.
 21. The electrospray device according to claim 20, wherein therepositioning comprises receding the conduit away from one entranceorifice, positioning the conduit in line with another entrance orifice,and placing the conduit in sealing engagement with the new entranceorifice to provide fluid to the new entrance orifice.
 22. Theelectrospray device according to claim 15, wherein the plurality offirst and second electrode pairs corresponding to a respective one ofeach of the plurality of exit orifices are controlled independently ofone another to provide a selected voltage to each exit orifice.
 23. Theelectrospray device according to claim 14, further comprising aplurality of conduits each positioned to provide fluid to a respectiveone of each of the plurality of entrance orifices.
 24. A system forprocessing droplets/sprays of fluid comprising: an electrospray deviceaccording to claim 14; and a daughter plate having a plurality of fluidreceiving wells at least one positioned to receive fluid droplets/sprayejected from a respective one of the plurality of exit orifice.
 25. Asystem for processing droplets/sprays of fluid comprising: anelectrospray device according to claim 14; and a mass spectrometrydevice having a plurality of ion sampling orifices at least onepositioned to receive droplets/sprays of fluid ejected from a respectiveone of the plurality of exit orifices.
 26. A system for processingdroplets/sprays of ink comprising: an ink jet printer which comprises anelectrospray device according to claim 14, that ejects droplets/spraysof ink from the plurality of exit orifices.
 27. A system for processingdroplets/sprays of fluid comprising: an electrospray device according toclaim 14; and a device to provide fluid to the plurality of entranceorifices of the electrospray device.
 28. A method of liquid separationanalysis comprising: providing the system according to claim 27, wherethe device to provide fluid to the entrance orifice of said electrospray device is a liquid separation device, wherein said methodcomprises: passing a fluid through the liquid separation device so thatthe fluid is subjected to liquid separation analysis and passing a fluidinto the entrance orifice, through the channel, and through the exitorifice under conditions effective to produce an electrospray.
 29. Themethod according to claim 28, wherein the liquid separation analysis isselected from the group consisting of capillary electrophoresis,capillary dielectrophoresis, capillary electrochromatography, and liquidchromatography.
 30. The electrospray device according to claim 1,further comprising: a nozzle positioned between the exit orifice and therecess, wherein the nozzle has a cross-sectional area of less than about3000 μm².
 31. The electrospray device according to claim 1, wherein thenozzle has an inner diameter of up to 50 μm.
 32. The electrospray deviceaccording to claim 1, wherein the nozzle has an inner diameter of up to30 μm.
 33. The electrospray device according to claim 1, wherein thenozzle has an inner diameter of from 2 μm to 50 μm.
 34. The electrospraydevice according to claim 1, wherein the nozzle has an inner diameter offrom 2 μm to 30 μm.
 35. The electrospray device according to claim 1,wherein the nozzle has an inner diameter of 15 μm.
 36. The electrospraydevice according to claim 1, wherein the nozzle has an inner diameter of5 μm.
 37. The electrospray device according to claim 1, wherein thenozzle has an inner diameter of 2 μm.
 38. The electrospray deviceaccording to claim 1, wherein the nozzle has an outer diameter of up to60 μm.
 39. The electrospray device according to claim 1, wherein thenozzle has an outer diameter of up to 40 μm.
 40. The electrospray deviceaccording to claim 1, wherein the nozzle has an outer diameter of 20 μm.41. The electrospray device according to claim 1, wherein the nozzle hasan outer diameter of 5 μm.
 42. The electrospray device according toclaim 1, wherein the nozzle has an outer diameter of from 5 to 60 μm.43. A system for processing droplets/sprays of fluid comprising: anelectrospray device according to claim 1, and a mass spectrometry deviceto receive droplets/sprays of fluid from the exit orifice of theelectrospray device.
 44. A method of mass spectrometric analysiscomprising: providing the system according to claim 43, wherein thedevice to receive fluid droplets/sprays of fluid from the exit orificeof said electrospray device is a mass spectrometer, wherein said methodcomprises: passing a fluid into the entrance orifice, through thechannel, and through the exit orifice under conditions effective toproduce an electrospray and passing the electrospray into the massspectrometer, whereby the fluid is subjected to a mass spectrometryanalysis.
 45. A system for processing droplets/sprays of fluidcomprising: an electrospray device according to claim 1; and a conduitpositioned to direct fluid into the entrance orifice.
 46. The systemaccording to claim 45, where the conduit contains a plurality of spacedapart posts.
 47. The system according to claim 46, wherein the pluralityof posts are spaced apart by no more than 2 μm.
 48. The system accordingto claim 46, wherein the posts have an outer coating of an insulatingmaterial.
 49. The system according to claim 48, wherein the insulatingmaterial is selected from the group consisting of silicon dioxide,silicon nitride, and combinations thereof.
 50. The system according toclaim 45, wherein the substrate further comprises: a fluid reservoir influid communication with the fluid passage; a fluid inlet to directfluid entering said substrate into the fluid reservoir; and a fluidoutlet to direct fluid from the fluid passage to the entrance orifice ofsaid electrospray device.
 51. The system according to claim 50, whereinthe substrate has opposed first and second surfaces with the fluidreservoir and the fluid passage being depressions in the first surfaceof the substrate, said system further comprising: a second substratejoined to the first surface of the substrate to cover the fluidreservoir and the fluid passage.
 52. The system according to claim 51,wherein the fluid inlet and the fluid outlet extend through the surfaceof the substrate.
 53. The system according to claim 50, wherein thesubstrate comprises a plurality of fluid passages, a plurality of fluidreservoirs each in fluid communication with one of the plurality fluidpassages, a plurality of fluid inlets to direct fluid entering saidsecond substrate into one of the plurality of fluid reservoirs, and aplurality of fluid outlets to direct fluid from the fluid passages to anentrance orifice of said electrospray device.
 54. The system accordingto claim 50, wherein the substrate has opposed first and second surfacesand the device to provide fluid comprises: a second substratecomprising: a fluid reservoir in fluid communication with the fluidpassage; a fluid inlet to direct fluid entering said second substrateinto the fluid reservoir; and a fluid outlet to direct fluid from thefluid passage to the entrance orifice of said electrospray device,wherein substrate is joined to the second substrate to cover the fluidreservoir and the fluid passage.
 55. A method of generating anelectrospray comprising: providing an electro spray device according toclaim 1; passing a fluid into the entrance orifice, through the channel,and through the exit orifice; applying a first potential to the firstelectrode; and applying a second potential to the second electrode,whereby fluid discharged from the exit orifice forms an electrospray.56. The method according to claim 55, wherein the electrospray is in theform of droplets.
 57. The method according to claim 55, wherein theelectrospray is in the form of a spray.
 58. The method according toclaim 55, further comprising: detecting components of the electrosprayby spectroscopic detection.
 59. The method according to claim 58,wherein the spectroscopic detection is selected from the groupconsisting of UV absorbance, laser induced fluorescence, and evaporativelight scattering.
 60. A method of mass spectrometric analysiscomprising: providing an electrospray device according to claim 1;providing a liquid separation device to provide fluid to the entranceorifice of said electrospray device; providing a mass spectrometer toreceive fluid droplets/sprays of fluid from the exit orifice of saidelectrospray device; passing a fluid through the liquid separationdevice so that the fluid is subjected to liquid separation analysis;passing a fluid into the entrance orifice, through the channel, andthrough the exit orifice under conditions effective to produce anelectrospray; and passing the electrospray into the mass spectrometer,whereby the fluid is subjected to a mass spectrometry analysis.
 61. Themethod according to claim 60, wherein the liquid separation analysis isselected from the group consisting of capillary electrophoresis,capillary dielectrophoresis, capillary electrochromatography, and liquidchromatography.
 62. A method for processing multiple sprays of fluidcomprising: providing an electrospray device according to claim 1;providing a device to provide at least one fluid sample to at least oneentrance orifice of said electrospray device; providing a device toreceive multiple sprays of fluid or droplets from said electrospraydevice; passing a fluid from said fluid providing device to saidelectrospray device; generating an electric filed surrounding the exitorifice of said at least one spray unit such that fluid dischargedtherefrom forms an electrospray or droplets; and passing saidelectrospray or droplets from said electrospray device to said receivingdevice.
 63. The method of claim 62 further comprising using saidreceiving device for performing mass spectrometry analysis; liquidchromatography analysis; protein, DNA, or RNA, combinatorial chemistryanalysis; proteomic screening; and ink jet printing.
 64. A system forprocessing droplets/sprays of fluid comprising: an electrospray devicecomprising: a substrate having an injection surface and an ejectionsurface opposing the injection surface, wherein the substrate comprises:an entrance orifice on the injection surface; an exit orifice on theejection surface; a channel extending between the entrance orifice andthe exit orifice; and a recess extending into the ejection surface andsurrounding the exit orifice; and a device to provide fluid to theelectrospray device comprising: a fluid passage; a fluid reservoir influid communication with the fluid passage; a fluid inlet to directfluid entering the device into the fluid reservoir; and a fluid outletto direct fluid from the fluid passage to the entrance orifice of saidelectrospray device, wherein the cross-sectional area of the entranceorifice of said electrospray device is equal to or less than thecross-sectional area of the fluid passage.
 65. A system according toclaim 64, wherein the ratio of the exit orifice inner cross-sectionalarea to the fluid passage cross-sectional area is less than about
 2. 66.A system according to claim 64, wherein the exit orifice innercross-sectional area is substantially equal to the fluid passagecross-sectional area.